Chemistry

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UNIT - I LESSON 1 THEORY OF ATOMIC AND MOLECULAR ORBITALS

Contents: 1.0 Aims and Objectives 1.1 Introduction 1.2 Theory of Atomic Orbital 1.2.1. Hybridization theory of atomic orbitals 1.2.2 Hybridization theory and the VSEPR approach 1.2.3 Hybridization theory vs. MO theory 1.3 Let us Sum Up 1.4 Lesson End Activities 1.5 Check your Progress 1.4 References
1.0 AIMS AND OBJECTIVES

v To describe, chemical bonding using descriptive atomic and molecular orbital theory; v To understand how atomic orbitals may be combined to give bonding, nonbonding and anti-bonding molecular orbitals. v To know about the Hybridization of Atomic orbitals v To predict hybridization of the central atom using the molecular profiles of the VSEPR Approach
1.1 INTRODUCTION
By sharing electron, molecules can form bonds, and it is possible to regard the sharing of two electrons by two atoms as constituting a chemical bond. Atoms can share one, two or three electrons (forming single, double and triple bonds). A hydrogen atom consists of a nucleus (a proton) and an electron. It is not possible to accurately determine the position of the electron, but it is possible to calculate the probability of finding the electron at any point around the nucleus. With hydrogen atom the probability distribution is spherical around the nucleus and it is possible to draw a spherical boundary surface, inside which there is a 95% possibility of finding the electron. The electron has a fixed energy and a fixed spatial distribution called an orbital. In the helium atom there are two electrons
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associated with the helium nucleus. The electrons have the same spatial distribution and energy (i.e. they occupy the same orbital), but they differ in their spin (Pauli exlusion principle). In general, electrons in atomic nuclei occupy orbitals of fixed energy and spatial distribution, and each orbital only contains a maximum of two electrons with antiparallel spins. In physics, periodic phenomena are associated with a "wave equation", and in atomic theory the relevant equation is called the "Schrdinger Equation". The wave equation predicts discrete solutions in one dimension for a particle confined to a box with infinite walls, the solutions can be shown as in the figure below:
Fig 1: Discrete solutions of wave equations.
y1 - y4 represent solutions of increasing energy. In three dimensions, the equation determines the energy and defines the spatial distribution of each electron. Solutions of the wave equations in three-dimensions allow calculation of the "shape" of each orbital.
Fig 2: The first five solutions of the wave equation for an electron associated with a proton
In the hydrogen atom, the 1s atomic orbital has the lowest energy, while the remainder (2s, 2px, 2py and 2pz) are of equal energy (ie.degenerate), but for all other atoms, the 2s atomic orbital is of lower energy than the 2px, 2py and 2pz orbitals, which are degenerate. In atoms, electrons occupy atomic orbitals, but in molecules they occupy similar molecular orbitals, which surround the molecule. The simplest molecule is hydrogen, which can be considered to be made up of two separate protons and electrons. There are two molecular orbitals for hydrogen; the lower energy orbital has its greater electron density between the two nuclei. This is the bonding molecular orbital - and is of lower energy than the two 1s atomic orbitals of hydrogen atoms making this orbital more stable than two separated atomic hydrogen orbitals. The upper molecular orbital has a node in the electronic wave function and the electron density is low between the two positively charged nuclei. The energy of the upper orbital is greater than that of the 1s atomic orbital and such an orbital is called an antibonding molecular orbital. Normally, the two electrons in hydrogen occupy the bonding molecular orbital, with antiparallel spins. If molecular hydrogen is irradiated by ultra-violet (UV) light, the molecule may absorb the energy, and promote one electron into its antibonding orbital (s* ), and the atoms will separate. The energy levels in a hydrogen molecule can be represented in a diagram - showing how the two 1s atomic orbitals combine to form two molecular orbitals, one bonding (s) and one antibonding (s* ).
Fig 3: Energy levels in a molecule
1.2 THEORY OF ATOMIC ORBITAL

This approach is also called the Atomic Orbital Approach to Bonding. The basic premise of this theory is that bonds are formed when atoms get close enough so that atomic orbitals on the individual atoms will be able to overlap so that the three dimensional probability regions share a common volume. This effectively increases the probability of finding bonding electrons between the two atoms. It also effectively results in the lowering of the energy state of the molecular system making the molecule more stable as a result of the overlap. The greater the overlap, the greater is the strength of the bond.
Pure Atomic Orbital Overlap The simplest of these bonds involve the overlap of two "s" orbitals as in the example when two Hydrogen atoms get close enough to bond. The "s" orbitals overlap to form a "sigma" bond between the two "s" orbitals. A second type of overlap is between two "p" orbitals to form a sigma bond between two "p" orbitals. An example is the p-p overlap between two Chlorine atoms. A third type of sigma overlap is the overlap between an "s" orbital and a "p" orbital such as when a Hydrogen atom's "s" orbital overlaps with a "p" orbital of another atom like a Chlorine atom. 1.2.1. Hybridization Theory of Atomic Orbitals One very powerful theory in the Valence Bond Approach is the Hybridization Theory which helps to explain why carbon containing molecules have carbon with four bonds. According to the orbital diagram of a ground state normal carbon atom, there are only two unpaired valence electrons in the 2p orbitals of a carbon atom. This should result in a carbon atom only capable of forming two bonds. However, every neutral carbon atom is tetra- valent, and therefore, should have four unpaired electrons from which to form four bonds. How can we account for the discrepancy, and how do we explain the tetra- valency of carbon atoms? Professor Linus Pauling from California Institute of Technolgy in California suggested an interesting theory to explain the discrepancy. The theory also offers an explaination why carbon containing molecules can have one of three geometries around each carbon atom in the molecule. This hybridization theory resulted in Pauling being awarded the Nobel Prize for Chemistry. According to this theory, carbon atoms are capable of hybridizing the s and p valence orbitals in one of three different ways. This hybridization process is preceded by the formation of an excited state carbon atom where a 2s electron is promoted into a 2p orbital before the hybridization process begins. Hybridization is similar to the hybridizations that occur in the plant and animal kingdoms. This results in a hybrid species which has some of the characteristics of both parents. The 2s and 2p orbitals of the excited state carbon can form one of three types of hybridized atomic orbitals. All four partially filled orbitals (one 2s and three 2p orbitals) may undergo mixing or hybridization to form four equal energy hybridized orbitals referred to as sp3 hybrid orbitals. Each of the four sp3 orbitals has an unpaired electron explaining the tetravalency for such an sp3 carbon. The four sp3 hybrid orbitals are arranged around the nucleus of the sp3 hybridized carbon atom with the orbitals pointing toward the corners of a tetrahedron. The angle separation between the hybrid orbitals is 109 degrees. In order for other atoms to effectively overlap their orbitals with the sp3 orbitals the atoms have to assume the same tetrahedral orientation. This results in an sp3 carbon atom forming four single covalent bonds. If a carbon atom has four single bonds around it, you can be sure that it is hybridized sp3 . Another type of hybridization involves only the mixing of three of the orbitals (one 2s and two 2p orbitals). This forms three hybrid orbitals around the carbon nucleus called
with one pure 2p orbital remained unhybridized. Each of the sp2 hybrid orbitals and the pure p orbital have an unpaired electron which accounts for the tetra-valency of an sp2 carbon. The sp2 orbitals around an sp2 hybridized carbon will have these orbitals pointing towards the corners of an equilateral triangle with the hybrid orbitals in the same plane as the carbon nucleus. The sp2 hybrid orbitals will be 120 degree separated. This orientation of the hybrid orbitals establishes a trigonal planar orientation. This means that in order for other atoms to form effective maximum overlap with an sp2 hybridized atom these atoms must orient in the same geometrical orientation. These three orbitals can overlap with three other orbitals to account for three bonds, but what happened to the fourth bond? We are forgetting the 2p orbital that did not undergo hybridization. This double lobed orbital will be perpendicular to the plane where the hybrid orbitals are. This p orbital can overlap with another p orbital from another sp2 hybrid carbon or from an Oxygen atom. This in effect makes for the second bond between the two atoms hence a double co-valent bond. The second bond is referred to as a "Pi" bond while the overlap between two sp2 orbitals between the two carbons is called a sigma bond. Pi bonds are considerably weaker than any sigma bond which accounts for the fact that Pi bonds are the first to be broken during a chemical reaction. The breaking of the Pi bond makes available unpaired electrons which can be shared by other incoming atoms. These are quite understandably known as addition type reactions. In essence, if there is a double bond attached to the carbon, that carbon will be sp2 hybridized. A third way that carbon can hybridize its orbitals is the mixing of only two of the orbitals (one 2s and one 2p orbitals). This forms two hybrid orbitals known as sp orbitals with two pure 2p orbitals left unhybridized. Again each of the two sp hybrid orbitals and the two pure 2p orbitals each have an unpaired electron to account for the tetra-valency of an sp hybrid carbon. The sp hybrid orbitals are oriented in a linear fashion with the hybrid orbitals 180 degrees separated. The two p orbitals are perpendicular to the linear arrangement of the sp hybrid orbitals and perpendicular to each other. Each p orbital can overlap with another p orbital orbital from another sp hybrid atom to form a Pi bond for each. This would mean that the two sp hybrid atoms have a sigma bond (overlap between the sp orbitals) and two Pi bonds (overlap of the p orbitals) or a total of three bonds. Any carbon that has a triple bond to it will be sp hybridized. This theory is specially designed for carbon chemistry, but it wouldn't be as powerful as it is it not for the fact that most other atoms can undergo hybridization. For example Boron atoms can undergo sp2 hybridization resulting in three sp2 hybrid orbitals and a pure 2p orbital remaining. The difference between a sp2 hybrid carbon and a sp2 hybrid Boron is that the Boron atom having one less valence electron has no electron in the pure 2p orbital that didn't enter into the hybridization process. Boron compounds have the same geometry as would be expected around the sp2 hybrid carbon but the 2p orbital would be empty. That accounts for the Boron compounds acting as Lewis acids which accept electrons. Aluminum compounds exhibit this same sp2 hybridization tendency as Boron. Silicon containing compounds find that Silicon atoms can hybridize sp3 which accounts for the similar geometry between CH4 and SiH4
According to the hybridization theory, the hybridized state of an atom can undergo transformation during a chemical reaction. For example: CH2 =CH2 + H2 -----> CH3 -CH3 . . . (1) The above reaction involves the adding of two Hydrogen atoms to the double bonded compound. The hybridization of the two carbons in the reactant is sp 2 . However the hybridization of the two carbon atoms in the product molecule is sp 3 . Obviously, the carbons were changed from sp2 to sp3 hybridized state. This phenomenon is reversible since the CH3 -CH3 compound can loose two Hydrogen atoms to produce a double bond. Another example is as follows: BF3 + F- ------> BF4 . . . . (2) In the above reaction the Hybridization of the Boron in the BF3 is clearly sp2 , but the BF4 ion product can be shown to be sp3 hybridized. Hybridization theory can also account for the fact that a carbon-carbon single bond length is longer (1.54 Angstroms) than the carbon=carbon double bond length (1.31 Angstroms) which in turn is longer than the carbon carbon triple bond length (1.2 Angstrom). This tendency in bond length can be explained by using Hybridization Theory. Carbon-Carbon single bonds involve sp3 carbons. If we look at the characteristics of an sp3 orbital we find that it is made out of the mixing of one s orbital which is characteristically less extended than p orbitals with three p orbitals which are more extended. We could say that the % of "s" character is 25% (1/4 of the orbitals used in the hybridization process are s). Using the same reasoning the sp2 orbital is 33% s in character (1/3 of the orbitals used in the hybridization process are s). For the sp hybrid orbitals the percentage of s character is 50% (1/2 of the orbitals used in the hybridization process are s). Since the sp3 orbitals that form a single bond between two carbons have the lowest percentage of s character you would expect their orbitals to be the most extended. That means that the overlap of sp3 orbitals by the carbon atoms can be effectively made when the nucleii are relatively far apart which would explain the relatively longer bond. The double bond between two sp2 carbons would mean that the orbitals that needed to overlap would involve a higher percentage of s character which means that the hybrid orbitals would be relatively less extended compared to sp3 orbitals. As a result the distance between the nuclei can be further apart for effective overlap to occur. A triple bond between two sp hybrid carbons means that the orbitals having the highest percentage of s character will be the orbitals least extended from the nucleii. As a result, the overlap between two sp hybrid carbons can not be effectively completed unless the nucleii of the two atoms are relatively close together. Hence the triple bond is the shortest. 1.2.2 Hybridization Theory and the VSEPR Approach In order to predict the hybridization of the central atom using the molecular profiles of the VSEPR Approach, one has to deal with the electron pair geometry. This will be different then the molecular geometry. Molecular geometry considers the shape with the atoms being considered alone. Electron pair geometry considers any non-bonding electron pairs as groups as well in determining the electron pair geometry. For example,
AB4 profile would be tetrahedral molecular geometry. Since there are no non-bonding pairs on the central atom, the electron pair geometry would be the same. In fact all molecular profiles in which there are zero non-bonding (or lone) pairs on the central atom would have the same geometry for molecular geometry and non-bonding pair geometry. 1. AB6 = octahedral = molecular geometry = non-bonding electron pair geometry = sp3 d2 hybridization of central atom 2. AB5 = Trigonal Bipyramidal = molecular geometry = non-bonding electron pair geometry = sp3 d hybridization on the central atom 3. AB4 = Tetrahedral = molecular geometry = non-bonding electron geometry = sp3 hybridization on central atom 4. AB3 = Trigonal Planar = molecular geometry = non-bonding electron pair geometry = sp2 hybridization of the central atom 5. AB2 = linear = molecular geometry = non-bonding electron pair geometry = SP hybridization on the central atom For molecular profiles where there is one or more non-bonding electron pairs attached to the central atom the non-bonding electron pair geometry which decides the hybridization of the central atom will differ from the molecular geometry where non-bonding electron pairs are not counted. 1. :AB2 = non-bonding electron pair geometry = trigonal planar = sp2 hybridization on the central atom 2. :AB3 = ::AB2 = non-bonding electron pair geometry = tetrahedral = sp3 hybridization of central atom 3. :AB4 = ::AB3 = :::AB2 = non-bonding electron pair geometry = Trigonal Bipyramidal = sp3 d hybridization of the central atom 4. :AB5 = ::AB4 = non-bonding electron pair geometry = octahedral = sp3 d2 hybridization of central atom 1.2.3 Hybridisation theory vs. MO theory Hybridisation theory is an integral part of organic chemistry and in general discussed together with molecular orbital theory in advanced organic chemistry textbooks although for different reasons. One textbook notes that for drawing reaction mechanisms sometimes a classical bonding picture is needed with 2 atoms sharing two electrons. It also comments that predicting bond angles in methane with MO theory is not straightforward. Another textbook treats hybridisation theory when explaining bonding in alkenes and a third uses MO theory to explain bonding in hydrogen but hybridisation theory for methane. Although the language and pictures arising from Hybridisation Theory, more widely known as Valence Bond Theory, remain widespread in synthetic organic chemistry, this qualitative analysis of bonding has been largely superseded by molecular orbital theory in other branches of chemistry. For example, inorganic chemistry texts have all but abandoned instruction of hybridisation, except as a historical footnote.One specific problem with hybridisation is that it incorrectly predicts the photoelectron spectra of many molecules, including such fundamental species such as methane and water. From a pedagogical perspective, hybridisation approach tends to over-emphasize localization of
bonding electrons and does not effectively embrace molecular symmetry as does MO Theory.
1.3 LET US SUM UP

In this lesson, we: Described the terms wave equations and Schrdinger equations and how these equations are implemented to predicts discrete solutions in one dimension for a particle confined to a box with infinite walls. Described the terms Bonding and Anti - bonding Bonds are formed when atoms get close enough so that atomic orbitals on the individual atoms will be able to overlap so that the three dimensional probability regions share a common volume. This effectively increases the probability of finding bonding electrons between the two Solutions of the wave equations in three-dimensions allow calculation of the "shape" of each orbital.
1.4 LESSON END ACTIVITIES

1. Highlight the features of hybridization theory of atomic orbitals. 2. Hybradization theory vs VSPER Discuss.
1.5 CHECK YOUR PROGRESS

1. Review the wave equations and schrodinger equations. 2. Define and highlight the effects of bonding and anti-bonding.
1.6 REFERENCES
Michael Edenborough
Jonathan Clayden, Nick Greeves, Stuart Warren, and Peter Wothers Marye Anne Fox James K. Whitesell
Organic Reaction Mechanisms Organic Chemistry Organic Chemistry Modern Inorganic Chemistry Computational Quantum Chemistry
W.L. Jolly Charles M. Quinn
http://www.chem.purdue.edu/gchelp/vsepr/ http://en.wikipedia.org/wiki/VSEPR_theory
LESSON: 2 MOLECULAR ORBITAL THEORY

Contents: 2.0 Aims and Objectives 2.1 Introduction 2.2 Forming molecular orbitals 1.2.1 Using the molecular orbital model to explain why some molecules do not exist 1.2.2 Molecular orbitals of the second energy level 2.3 Bond Order 2.4 Orbitals for Selected Molecules 2.4.1 Saturated molecules 2.4.2 Molecules with double bonds 2.4.3 Molecules with electron lone pairs 2.4.4 Conjugated and aromatic molecules 2.5 Let us Sum Up 2.6 Lesson End Activities 2.7 Check your Progress 2.8 References

In this lesson, we Have discussed the molecular orbital theory and formation of molecular orbitals. Explained Why Some Molecules Do Not Exist Using the Molecular Orbital Model Explained how to calculate bond orders from Lewis structures
2.1 INTRODUCTION
Molecular orbital theory (MO theory) is a method for determining molecular structure in which electrons are not assigned to individual bonds between atoms, but are treated as moving under the influence of the nuclei in the whole molecule. In this theory, each molecule has a set of molecular orbitals, in which it is assumed that the molecular orbital
wave function f may be written as a simple weighted sum of the n constituent atomic orbitals i, according to the following equation
j =
. . . (1)
The cij coefficients may be determined numerically by substitution of this equation into the Schrdinger equation and application of the variational principle. This method is called the linear combination of atomic orbitals approximation and is used in computational chemistry. An additional unitary transformation can be applied on the system to accelerate the convergence in some computational schemes. Molecular orbital theory was seen as a competitor to valence bond theory in the 1930s, before it was realised that the two methods are closely related and that when extended they become equivalent.
2.2 FORMING MOLECULAR ORBITALS

Molecular orbitals are obtained by combining the atomic orbitals on the atoms in the molecule. Consider the H2 molecule, for example. One of the molecular orbitals in this molecule is constructed by adding the mathematical functions for the two 1s atomic orbitals that come together to form this molecule. Another orbital is formed by subtracting one of these functions from the other, as shown in the figure below.
Fig 4: Formation of molecular orbitals of H2 molecule.
One of these orbitals is called a bonding molecular orbital because electrons in this orbital spend most of their time in the region directly between the two nuclei. It is called a sigma ( ) molecular orbital because it looks like an s orbital when viewed along the H-H bond. Electrons placed in the other orbital spend most of their time away from the region between the two nuclei. This orbital is therefore an antibonding, o r sigma star ( *), molecular orbital.
Fig 5: Energy level of an H2 molecule
The bonding molecular orbital concentrates electrons in the region directly between the two nuclei. Placing an electron in this orbital therefore stabilizes the H2 molecule. Since the * antibonding molecular orbital forces the electron to spend most of its time away from the area between the nuclei, placing an electron in this orbital makes the molecule less stable. Electrons are added to molecular orbitals, one at a time, starting with the lowest energy molecular orbital. The two electrons associated with a pair of hydrogen atoms are placed in the lowest energy, or bonding, molecular orbital, as shown in the figure below. This diagram suggests that the energy of an H2 molecule is lower than that of a pair of isolated atoms. As a result, the H2 molecule is more stable than a pair of isolated atoms.
Fig 6 The status of the electrons of hydrogen atoms
2.2.1 Using the Molecular Orbital Model to Explain Why Some Molecules Do Not Exist This molecular orbital model can be used to explain why He2 molecules don't exist. Combining a pair of helium atoms with 1s2 electron configurations would produce a molecule with a pair of electrons in both the bonding and the * antibonding molecular orbitals. The total energy of an He2 molecule would be essentially the same as the energy of a pair of isolated helium atoms, and there would be nothing to hold the helium atoms together to form a molecule. The fact that an He2 molecule is neither more nor less stable than a pair of isolated helium atoms illustrates an important principle: The core orbitals on an atom make no contribution to the stability of the molecules that contain this atom. The only orbitals that are important in our discussion of molecular orbitals are those formed when valence-shell orbitals are combined. The molecular orbital diagram for an O2 molecule would therefore ignore the 1s electrons on both oxygen atoms and concentrate on the interactions between the 2s and 2p valence orbitals.
2.2.2 Molecular Orbitals of the Second Energy Level The 2s orbitals on one atom combine with the 2s orbitals on another to form a 2s bonding and a 2s* antibonding molecular orbital, just like the 1s and 1s* orbitals formed from the 1s atomic orbitals. If we arbitrarily define the Z axis of the coordinate system for the O2 molecule as the axis along which the bond forms, the 2pz orbitals on the adjacent atoms will meet head-on to form a 2p bonding and a 2p * antibonding molecular orbital, as shown in the figure below. These are called sigma orbitals because they look like s orbitals when viewed along the oxygen-oxygen bond.
Fig 7: Formation of Sigma orbitals
The 2px orbitals on one atom interact with the 2px orbitals on the other to form molecular orbitals that have a different shape, as shown in the figure below. These molecular orbitals are called pi ( ) orbitals because they look like p orbitals when viewed along the bond. Whereas and * orbitals concentrate the electrons along the axis on which the nuclei of the atoms lie, and * orbitals concentrate the electrons either above or below this axis.
Fig 8: Formation of Pi orbitals
The 2px atomic orbitals combine to form a x bonding molecular orbital and a x * antibonding molecular orbital. The same thing happens when the 2py orbitals interact, only in this case we get a y and a y * antibonding molecular orbital. Because there is no difference between the energies of the 2px and 2py atomic orbitals, there is no difference between the energies of the x and y or the x * and y * molecular orbitals. The interaction of four valence atomic orbitals on one atom (2s, 2px , 2py and 2pz) with a set of four atomic orbitals on another atom leads to the formation of a total of eight molecular orbitals: 2s, 2s*, 2p , 2p *, x , y , x *, and y *. There is a significant difference between the energies of the 2s and 2p orbitals on an atom. As a result, the 2s and *2s orbitals both lie at lower energies than the 2p , 2p *, x , y , x *, and y * orbitals. To sort out the relative energies of the six molecular orbitals formed when the 2p atomic orbitals on a pair of atoms are combined, we need to understand the relationship between the strength of the interaction between a pair of orbitals and the relative energies of the molecular orbitals they form. Because they meet head-on, the interaction between the 2pz orbitals is stronger than the interaction between the 2px or 2py orbitals, which meet edge-on. As a result, the 2p orbital lies at a lower energy than the x and y orbitals, and the 2p * orbital lies at higher energy than the x * and y * orbitals, as shown in the figure below.
Fig 9 The models describing O2 and F2
Unfortunately an interaction is missing from this model. It is possible for the 2s orbital on one atom to interact with the 2pz orbital on the other. This interaction introduces an element of s-p mixing, or hybridization, into the molecular orbital theory. The result is a slight change in the relative energies of the molecular orbitals, to give the diagram shown in the figure below. Experiments have shown that O2 and F2 are best described by the model in the figure above, but B2 , C2 , and N2 are best described by a model that includes hybridization, as shown in the figure below Fig. 10.
Fig 10 The models described for B2 , C2 , N2
2.3 BOND ORDER

The number of bonds between a pair of atoms is called the bond order. Bond orders can be calculated from Lewis structures, which are the heart of the valence-bond model. Oxygen, for example, has a bond order of two.
When there is more than one Lewis structure for a molecule, the bond order is an average of these structures. The bond order in sulfur dioxide, for example, is 1.5 the average of an S-O single bond in one Lewis structure and an S=O double bond in the other.
In molecular orbital theory, we calculate bond orders by assuming that two electrons in a bonding molecular orbital contribute one net bond and that two electrons in an antibonding molecular orbital cancel the effect of one bond. We can calculate the bond order in the O2 molecule by noting that there are eight valence electrons in bonding molecular orbitals and four valence electrons in antibonding molecular orbitals in the electron configuration of this molecule. Thus, the bond order is two.
. . . (2) Although the Lewis structure and molecular orbital models of oxygen yield the same bond order, there is an important difference between these models. The electrons in the Lewis structure are all paired, but there are two unpaired electrons in the molecular orbital description of the molecule. As a result, we can test the predictions of these theories by studying the effect of a magnetic field on oxygen. Atoms or molecules in which the electrons are paired are diamagnetic repelled by both poles of a magnetic. Those that have one or more unpaired electrons are
paramagnetic attracted to a magnetic field. Liquid oxygen is attracted to a magnetic field and can actually bridge the gap between the poles of a horseshoe magnet. The molecular orbital model of O2 is therefore superior to the valence-bond model, which cannot explain this property of oxygen.
2.4 ORBITALS FOR SELECTED MOLECULES

This section illustrates pictorially molecular orbitals for several organic and inorganic molecules. 2.4.1 Saturated molecules These are molecules in which all valence electrons are involved in the formation of single bonds. There are no non-bonded lone pairs. These molecules are generally less reactive than either electron-rich or electron-deficient species, with all occupied orbitals having relatively low energies. Methane: The valence molecular orbitals of methane are delocalized over the entire nuclear skeleton - that is, it is not easy to assign any one orbital to a particular C-H bond. It is possible to see how complex the orbital structure becomes with the increase in energy. Methane has four valence molecular orbitals (bonding), consisting of one orbital with one nodal plane (lowest occupied) and three degenerate (equal energy) orbitals that do have a nodal plane.
Fig 11: Energy diagram and pictorial view of the orbitals in methane
2.4.2 Molecules with double bonds In molecules where the number of bonding electron pairs exceeds the number of unions between atoms, the extra electrons occupy higher energy molecular orbitals than the orbitals found in molecules where the number of bonding electron pairs equals the number of unions between atoms. These are double bonds, and the orbitals have a nodal plane containing the atoms sharing these p-type orbitals. Ethene: The simplest alkene is ethene. Its chemistry is dominated by two "frontier orbitals", that is the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO). For the ethene orbital energy diagram these are shown as p CC for the HOMO, and p * CC for the LUMO.An important property of the ethene molecule, and alkenes in general is the existence of a high barrier to rotation about the C=C which tends to hold the molecule flat.
Fig 12: The energy diagram and pictorial view of the orbitals in Ethene
2.4.3 Molecules with electron lone pairs Hydrogen Fluoride: A simple diatomic molecule is Hydrogen fluoride. There are eight valence electrons which occupy four molecular orbitals. The two highest energy MO's are degenerate, are p-type and have no electron density associated with the hydrogen atom, ie. they are Non-
Bonding Orbitals (NBO) and in Lewis Theory are represented as two "Lone Pairs". Another important difference between Hydrogen Fluoride and previous molecules is that the electron density is not equally distributed about the molecule. There is a much greater electron density around the fluorine atom. This is because fluorine is an exremely electronegative element, and in each bonding molecular orbital, fluorine will take a greater share of the electron density.
Fig 13 : Energy diagram and pictorial view of the orbitals in Hydrogen Fluoride
2.4.4 Conjugated and aromatic molecules p bonds in close proximity will often interact. Some of the delocalized molecular orbitals that result will be stabilized, while others will be destabilized. The individual combinations may be polarized, providing an increase in wave function amplitude on some centers at the expense of a decrease in amplitude on others. This gives rise to the possibility of more varied reactivity patterns than are observed for simple alkenes. Aromatic molecules exhibit a wide range of reactivity patterns toward both electron rich and electron deficient species. These mainly depend on the structures and energies of the frontier p-type molecular orbitals, the HOMO and LUMO. Except for non-bonded lone pairs, the s framework plays little role in the overall reactivity. Benzene: Benzene is the archetypal aromatic compound. It has a symmetrical p system and so is not over reactive on any one site. From the cyclic polyene diagram it can be seen that benzene has six p-molecular orbitals, (which contain the six p-electrons), three bonding and three anti-bonding. The upper bonding degenerate pair of orbitals are the HOMO's of benzene. The p orbital manifold is shown below - but also of interest is that the pattern of the p orbitals is repeated within the s system. The s functions - like the p orbitals are delocalized throughout the carbon skeleton. The six p electrons are arranged as in the fig. 14:
Fig 14 : A pictorial representation of the energy diagram for Benzene
Bonding in benzene From the above diagram it can be seen that the lowest lying orbital, p 1 , the orbital coefficients are such that the bonding character between each pair of adjacent carbon atoms is equal. In p 2 bonding only occurs between atoms C2 and C3 and between C5 and C6 since the coefficients on C1 and C4 are zero. In p 3 , C1 , which is bonded to C2 and C6 and C4 is bonded to C3 and C5 , there are anti-bonding interactions between C2 and C3 and between C 5 a n d C 6 . Therefore if we consider the pair of orbitals p 2 a n d p 3 the contribution to the C-C p bonding is equal for each bond. Since there are three occupied bonding orbitals and six CC linkages - the p bond order is 1 /2 . This description is in accord with the two resonating mesomeric forms (or Kekul structures in a) below in which single and double bond characters alternate around the ring. Conventionally, the Fig. 15 in b) is used to show that the six electrons are delocalized around the ring:
Fig 15 The position of electrons in a benzene ring
2.5 LET US SUM UP
Molecular orbitals are obtained by combining the atomic orbitals on the atoms in the molecule The electrons in the Lewis structure are all paired, but there are two unpaired electrons in the molecular orbital description of the molecule. This section illustrates pictorially molecular orbitals for several organic and inorganic molecules.
2.6 POINTS FOR DISCUSSION

1. How molecular orbital model helps using explaining why some molecular do not exist? 2. Elucidate the fact that helium molecule in neither more nor less stable than a pair of isolated heeling atoms.

Discuss the formation of Pi and Sigma orbitals.
2.8 REFERENCES
1. Daintith, J. (2004). Oxford Dictionary of Chemistry. New York: Oxford University Press. ISBN 0-19-860918-3. 2. Licker, Mark, J. (2004). McGraw-Hill Concise Encyclopedia of Chemistry. New York: McGraw-Hill. ISBN 0-07-143953-6. 3. Coulson, Charles, A. (1952). Valence. Oxford at the Clarendon Press.
L E S S O N 3 - LINEAR COMBINATION OF ATOMIC ORBITALS AND VALENCE BOND THEORY

Contents 3.0 Aims and Objectives 3.1 Linear Combination of Atomic Orbitals 3.2 Valence Bond Theory 3.3.1 Applications of VB theory 3.3 Let us Sum Up 3.4 Lesson End Activities 3.5 Check your Progress 3.6 References

In this lesson we have described about the linear combination of atomic orbitals and valence bond theory After reading this lesson you will be able to: Describe the calculation of molecular orbitals. Explains the nature of a chemical bond in a molecule in terms of atomic valencies using valence bond theory List applications of valence bond theory
3.1 LINEAR COMBINATION OF ATOMIC ORBITALS

A linear combination of atomic orbitals or LCAO is a quantum superposition of atomic orbitals and a technique for calculating molecular orbitals in quantum chemistry. In quantum mechanics, electron configurations of atoms are described as wavefunctions. In mathematical sense, these wave functions are the basis set of functions, the basis functions, which describe the electrons of a given atom. In chemical reactions, orbital wavefunctions are modified, i.e. the electron cloud shape is changed, according to the type of atoms participating in the chemical bond. It was introduced in 1929 by Sir John Lennard-Jones with the description of bonding in the diatomic molecules of the first main row of the periodic table, but had been used earlier by Pauling for H2 +.
A mathematical description is
=C1X1+C2X2+C3X3+. +CnXn
or
. . . (3)
. . . . (4)
where (phi) is a molecular orbital represented as the sum of the product of n atomic orbitals (chi) and n coefficients . The coefficients are the weights of the contributions of the n atomic orbitals to the molecular orbital. The orbitals are thus expressed as linear combinations of basis functions, and the basis functions are one-electron functions centered on nuclei of the component atoms of the molecule. The atomic orbitals used are typically those of hydrogen- like atoms since these are known analytically i.e. Slater-type orbitals but other choices are possible like Gaussian functions from standard basis sets. By minimizing the total energy of the system, an appropriate set of coefficients of the linear combinations is determined. This quantitative approach is now known as the Hartree-Fock method. However, since the development of computational chemistry, the LCAO method often refers not to an actual optimization of the wave function but to a qualitative discussion which is very useful for predicting and rationalizing results obtained via more modern methods. In this case, the shape of the molecular orbitals and their respective energies are deduced approximately from comparing the energies of the atomic orbitals of the individual atoms (or molecular fragments) and applying some recipes known as level repulsion and the like. The graphs that are plotted to make this discussion clearer are called correlation diagrams. The required atomic orbital energies can come from calculations or directly from experiment via Koopmans' theorem. This is done by using the symmetry of the molecules and orbitals involved in bonding. The first step in this process is assigning a point group to the molecule. A common example is water, which is of C2v symmetry. Then a reducible representation of the bonding is determined demonstrated in Fig. 16 for water:
Fig 16 : Representation of bonding in water molecule
Each operation in the point group is performed upon the molecule. The number of bonds that are unmoved is the character of that operation. This reducible representation is
decomposed into the sum of irreducible representations. These irreducible representations correspond to the symmetry of the orbitals involved.
3.2 VALENCE BOND THEORY

Valence bond theory explains the nature of a chemical bond in a molecule in terms of atomic valencies.Valence bond theory summarizes the rule that the central atom in a molecule likes to form electron pair bonds in accordance with geometric constraints as defined by the octet rule, approximately. Valence bond theory is closely related to molecular orbital theory. A valence bond structure is similar to a Lewis structure, however where a single Lewis structure cannot be written, several valence bond structures are used. Each of these VB structures represents a specific Lewis structure. This combination of valence bond structures is the main point of resonance theory. Valence bond theory considers that the overlapping atomic orbitals of the participating atoms form a chemical bond. Because of the overlapping, it is most probable that electrons should be in the bond region. Valence bond theory views bonds as weakly coupled orbitals (small overlap). Valence bond theory is typically easier to employ in ground state molecules. The overlapping atomic orbitals can differ. The two types of overlapping orbitals are sigma and pi. Sigma bonds occur when the orbitals of two shared electrons overlap headto-head. Pi bonds occur when two orbitals overlap when they are parallel. For example, a bond between two s-orbital electrons is a sigma bond, because two spheres are always coaxial. In terms of bond order, single bonds have one sigma bond, double bonds consist of one sigma bond and one pi bond, and triple bonds contain one sigma bond and two pi bonds. However, the atomic orbitals for bonding may be hybrids. Often, the bonding atomic orbitals have a character of several possible types of orbitals. The methods to get an atomic orbital with the proper character for the bonding is called hybridization. Valence bond theory now complements Molecular Orbital Theory (MO theory), which does not adhere to the VB idea that electron pairs are localized between two specific atoms in a molecule but that they are distributed in sets of molecular orbitals which can extend over the entire molecule. MO theory can predict magnetic properties in a straight forward manner, but valence bond theory is more complicated although giving the similar results. Valence bond theory views aromatic properties of molecules as due to resonance between Kekule, Dewar and possibly ionic structures, while molecular orbital theory views it as delocalisation of the -electrons. The underlying mathematics are also more complicated limiting VB treatment to relatively small molecules. On the other hand, VB theory provides a much more accurate picture of the reorganization of electronic charge that takes place when bonds are broken and formed during the course of a chemical reaction. In particular, valence bond theory correctly predicts the dissociation of homonuclear diatomic molecules into separate atoms, while simple molecular orbital theory predicts dissociation into a mixture of atoms and ions. More recently, several groups have developed what is often called modern valence bond theory. This replaces the overlapping atomic orbitals by overlapping valence bond orbitals that are expanded over all basis functions in the molecule. The resulting energies
are more competitive with energies where electron correlation is introduced based on a Hartree-Fock reference wavefunction. 3.3.1 Applications of VB theory An important aspect of the VB theory is the condition of maximum overlap which leads to the formation of the strongest possible bonds. This theory is used to explain the covalent bond formation in many molecules. For Example in the case of F2 molecule the F - F bond is formed by the overlap of p orbitals of the two F atoms each containing an unpaired electron. Since the nature of the overlapping orbitals are different in H2 and F2 molecules, the bond strength and bond lengths differ between H 2 and F2 molecules. In HF molecule the covalent bond is formed by the overlap pf 1s of H and 2p orbital of F each containing an unpaired electron. Mutual sharing of electrons between H and F results in a covalent bond between HF.
3.3 LET US SUM UP

In this lesson we have learnt How to calculate molecular orbital Nature of a chemical bond Applications of valence bond theory

How do the atomic orbital linearly combine and explain their effects. Valence bond theory explain the nature of a chemical bond clearly Justify.
3.5 REFERENCES
(1) Ahmed A. Hasanein, Myron W. Evans Chemistry Computational Methods in Quantum
(2) http://en.wikipedia.org/wiki/Valence_bond_theory (3) http://www.iupac.org/goldbook/VT07125.pdf
LESSON 4 - RESONANCE STRUCTURES

Contents 4.0 Aims and Objectives 4.1 Introduction 4.2 Drawing resonance structures 4.3 Let us Sum Up 4.4 Lesson End Activities 4.5 Check Your Progress 4.6 References

In this lesson we have described about the resonance structures of molecules. After reading this lesson you will be able to Describe the resonance structures of molecules Draw the resonance structures of various biomolecules
4.1 INTRODUCTION
Resonance can be considered as the condition ocurring when more than one valid Lewis structure can be written for a particular molecule. This can happen any time there are two or more adjacent p-tpye orbitals in the same plane. Resonance structures are imaginary. They represent extremes of electron location. Resonance can be considered as a valence theory solution to a molecular orbital problem. In general chemistry the concept of resonance was introduced through inorganic anions such as NO3-, NO2-, ClO4-, SO4-2 , CO3 -2 .
4.2 DRAWING RESONANCE STRUCTURES

There are some molecules for which we cannot write Lewis structures that agree with experimental observations. For example, the formate ion, CHO2 -, if drawn using Lewis rules, would be:
Fig 17 : Lewis structure of CHO2 -
But actually, experimental observation shows that the bond between the carbon and the two oxygens are the same length. Since a CO double bond is a different length then the single bond, the lewis structure must be wrong. Actually, observation shows that it is neither of the lengths of the single or double bond, but a resonance hybrid, and can be drawn like this:
Fig 18 : Resonance structure of CHO2 -
The bond is neither a double nor single bond. Both the double and single bond "contribute" to the entire structure. The double-ended arrow shows that we are drawing resonance structures and implies that the true hybrid is a composite of the two resonance structures. A simple way of determining whether resonance should be applied is that when you must move electrons to create one more double bonds, the number of resonance structures is equal to the number of equivalent choices for the locations of the double bonds. For example, In a nitrate ion, NO3 -, the double bond between N and the O can exist with any three of the oxygen. The resonance structures would then be:
Fig 19 : Resonance structure of NO3 -
The benefit of resonance hybrids in a molecule is that the total energy is lower than any one of its resonance structures, and therefore is more stable. A benzene molecule (see Organic Chemistry: Aromatic Hydrocarbons) is ring shaped and is drawn with alternating single or double bonds in between carbon atoms. It is a resonance hybrid though because the double and single bonds can alternate in a different fashion.
Fig 20: Resonance structure of Benzene.
The benzene ring is usually represented as a hexagon with a circle in the middle to show that the electron density of the three extra bonds is evenly distributed.
Fig 21. Benzene Ring
The extra stability achieved is called the resonance energy. It does not behave like other organic compounds with double bonds because breaking those double bonds would mean breaking the resonance hybrid, giving it more energy, and destabilizing it.
4.3 LET US SUM UP

Resonance structures are imaginary. They represent extremes of electron location. There are some molecules for which we cannot write Lewis structures that agree with experimental observations.

Justify the fact that resonance structures are imaginary.

Give a qualitative treatment on the resonance structures of NO3 .
4.6 REFERENCES
(1) Herbert Meislich, Jacob Sharefkin Organic Chemistry
(2) John C. Kotz et.al
Chemistry & Chemical Reactivity
(3) http://en.wikipedia.org/wiki/Resonance_ (chemistry)
LESSON-5 SIGMA BONDS AND PI BONDS

5.0 Aims and Objectives 5.1 Sigma bonds 5.1.1 Sigma bonds in polyatomic compounds 5.1.2 Sigma bonds in multiple bonded species 5.2 Pi Bonds ( Bonds) 5.3 Let us Sum Up 5.4 Lesson End Activities 5.5 Check your Progress 5.4 References

In this lesson we have described about the sigma bonds and pi bonds. Sigma bonds in polyatomic compounds and in multiple bonded species are explained. It is also described the significance of Pi bonds.
5.1 SIGMA BONDS

Sigma bonds ( bonds) are a type of covalent chemical bond. Sigma bonding is most clearly defined for diatomic molecules using the language and tools of symmetry groups. In this formal approach, a -bond is symmetrical with respect to rotation about the bond axis. By this definition, common forms of sigma bonds are s+s, pz+pz, and s+pz, and dz2+dz2 (where z is defined as the bond axis). Quantum theory also indicates that molecular orbitals (MO) of identical symmetry mix. As a practical consequence of mixing in diatomic molecules, the wavefunctions s+s and pz+pz molecular orbitals become blended. The extent of mixing (or blending) depends on the relative energies of the like-symmetry MO's.
Fig 22 : bond between two atoms : localization of electronic density.
For homodiatomics, bonding orbitals have no nodal planes between the bonded atoms. The corresponding antibonding, or * orbital, is defined by the presence of a nodal plane between these two bonded atoms. Sigma bonds are the strongest type of covalent bonds. Electrons in sigma bonds are sometimes referred to as sigma electrons. The symbol is the Greek letter for s. When viewed down the bond axis, a MO resembles an s atomic orbital.
5.1.1 Sigma bonds in polyatomic compounds The concept of sigma bonding is extended, albeit loosely, to describe bonding interactions involving overlap a single lobe of one orbital with a single lobe of another. For example, propane is described as consisting of ten sigma bonds, one each for the two C-C bonds and one each for the eight C-H bonds. The bonding in such a polyatomic molecule is highly delocalized, which conflicts with the two-orbital, one-bond concept. Despite this complication, the concept of bonding is extremely powerful and hence pervasive. 5.1.2 Sigma bonds in multiply bonded species Compounds that feature multiple bonds, such as ethylene and chromium(II) acetate have sigma bonds between the multiply bonded atoms. These sigma bonds are supplemented b y -bonds, e.g. in the case of ethylene, and even -bonds, e.g. in the case of chromium(II) acetate.
5.2 PI BOND ( bonds)

In chemistry, pi bonds ( bonds) are covalent nbgs where two lobes of one involved electron orbital overlap two lobes of the other involved electron orbital. Only one of the orbital's nodal planes passes through both of the involved nuclei. The Greek letter in their name refers to p orbitals, since the orbital symmetry of the pi bond is the same as that of the p orbital when seen down the bond axis. P orbitals usually engage in this sort of bonding. However, d orbitals can engage in pi bonding also. Pi bonds are usually weaker than sigma bonds because their (negatively charged) electron density is further from the positive charge of the atomic nucleus, which requires more energy. From the perspective of quantum mechanics, this bond weakness is explained by significantly less overlap between the component p-orbitals due to their parallel orientation. Although the pi bond by itself is weaker than a sigma bond, pi bonds are often components of multiple bonds, together with sigma bonds. The combination of pi and
sigma bond is stronger than either bond by itself. The enhanced strength of a multiple bond vs. a single (sigma bond) is indicated in many ways, but most obviously by a contraction in bond lengths. For example in organic chemistry, carbon-carbon bond lengths are ethane (154 pm), ethylene (133 pm) and acetylene (120 pm).
Fig 23 : Two p-orbitals forming a -bond.
In addition to one sigma bond, a pair of atoms connected via double bond and triple bonds have one or two pi bonds, respectively. Pi bonds result from overlap of atomic orbitals that with two areas of overlap. Pi-bonds are more diffuse bonds than the sigma bonds. Electrons in pi bonds are sometimes referred to as pi electrons. Molecular fragments joined by a pi bond cannot rotate about that bond without breaking the pi bond, because rotation involves destroying the parallel orientation of the constituent p orbitals.
Fig 24 : Pi bond in ethylene
In certain metal complexes, pi interactions between a metal atom and alkyne and alkene pi antibonding orbitals form pi-bonds. In some cases of multiple bonds between two atoms, there is no sigma bond at all, only pi bonds. Examples include diiron hexacarbonyl (Fe2(CO)6), dicarbon (C2) and the borane B2H2. In these compounds the central bond consists only of pi bonding, and in order to achieve maximum orbital overlap the bond distances are much shorter than expected.
5.3 LET US SUM UP

Sigma bonding is most clearly defined for diatomic molecules using the language and tools of symmetry groups. common forms of sigma bonds are s+s, pz+pz, and s+pz, and dz2+dz2. Sigma bonds are the strongest type of covalent bonds. These sigma bonds are supplemented by -bonds. Pi bonds are usually weaker than sigma bonds because their (negatively charged) electron density is further from the positive charge of the atomic nucleus, which requires more energy.

(1) Sigma bonds are strongest type of covalent bonds Justify. (2) Give reasons for Pi bonds being weaker than sigma bonds.

Make a comparitive study between sigma and pi bonds.
5.6 REFERENCES
(1) http://dl.clackamas.edu/ch106-02/sigma.htm (2) http://en.wikipedia.org/wiki/Sigma_bond (3) http://en.wikipedia.org/wiki/Pi_bond
LESSON 6- ELECTROVALENT BOND

Contents: 6.0 Aims and objectives 6.1 Introduction 6.2 Electrovalent Bond 6.2.1 Polarization effects 6.2.2 Ionic structure 6.2.3 Ionic versus covalent bonds 6.2.4 Electrical conductivity 6.2.5 Formation of Positive Ions 6.2.6 Formation of Negative Ions 6.3 Stability of electrovalent bond 6.3.1 Electron affinity: 6.3.2 Lattice energy 6.4 Let us Sum Up 6.5 Lesson End Activities 6.6 Check your Progress 6.7 Suggested Readings/References/Sources

In this unit we have described the electrovalent bond with their properties. After reading this unit, you should be able to Describe the nature of electrovalent bond To distinguish between an electrovalent bond and a covalent bond Describe electrical conductivity by electrovalent bond Describe the formation of positive ions and negative ions
6.1 INTRODUCTION
Atoms of almost every element have the ability to combine with other atoms to form more complex structures. The forces of attraction that bind them together are chemical
bonds. To understand chemistry, the nature and origin of chemical bonds is important, since the basis of chemical reactions is the forming and the breaking of bonds and the changes in bonding forces. There are two main classes of bonding forces: covalent bonds and ionic bonds. Covalent bonding deals with the sharing of electrons between atoms. Ionic bonding deals with the transfer of electrons between atoms. While the structure of ionic compounds are characterized by an orderly arrangement of its ions controlled primarily by the sizes of the ions and their charges and have no preferred directional properties, meaning that the array of ions can collapse is it is melted, the structure of molecular substances can be very different and complicated. Molecules have three-dimensional shapes determined by the relative orientations of their covalent bonds, which is maintained whether it is a solid, liquid, or gas. Properties of the molecule depend on the arrangement of the atoms. For example, enzymes, which make biochemical reactions happen faster, can lose their function if there are slight alterations in its structure, because they require precise fits between molecules.
6.2 ELECTROVALENT BOND

An ionic bond (or electrovalent bond) is a type of chemical bond based on electrostatic forces between two oppositely-charged ions. In ionic bond formation, a metal donates an electron, due to a low electronegativity to form a positive ion or cation. In ordinary table salt (NaCl), the bonds between the sodium and chloride ions are ionic bonds. Often ionic bonds form between metals and nonmetals. The non-metal atom has an electron configuration just short of a noble gas structure. They have high electronegativity, and so readily gain electrons to form negative ions or anions. The two or more ions are then attracted to each other by electrostatic forces. Ionic bonding occurs only if the overall energy change for the reaction is favorable when the bonded atoms have a lower energy than the free ones. The larger the resulting energy change the stronger the bond. Pure ionic bonding is not known to exist. All ionic bonds have a degree of covalent bonding or metallic bonding. The larger the difference in electro negativity between two atoms the more ionic the bond. Ionic compounds conduct electricity when molten or in solution. They generally have a high melting point and tend to be soluble in water. 1.2.1 Polarization effects Ions in crystal lattices of purely ionic compounds are spherical; however, if the positive ion is small and/or highly charged, it will distort the electron cloud of the negative ion. This polarization of the negative ion leads to a build-up of extra charge density between the two nuclei, i.e., to partial covalency. Larger negative ions are more easily polarized, but the effect is usually only important when positive ions with charges of 3+ (e.g., Al3 +) are involved (e.g., pure AlCl3 is a covalent molecule). However, 2+ ions (Be2 +) or even
1+ (Li+) show some polarizing power because their sizes are so small (e.g., LiI is ionic but has some covalent bonding present). 6.2.2 Ionic structure Ionic compounds in the solid state form a continuous ionic lattice structure in an ionic crystal. The simplest form of ionic crystal is a simple cubic. This is as if all the atoms were placed at the corners of a cube. This unit cell has a wht that is the same as 1 of the atoms involved. When all the ions are approximately the same size, they can form a different structure called a face-centered cubic (where the weight is 4 * atomic weight), but, when the ions are different sizes, the structure is often body-centered cubic (2 times the weight). In ionic lattices the coordination number refers to the number of connected ions. 6.2.3 Ionic versus covalent bonds In an ionic bond, the atoms are bound by attraction of opposite ions, whereas, in a covalent bond, atoms are bound by sharing electrons. In covalent bonding, the molecular geometry around each atom is determined by VSEPR rules, whereas, in ionic materials, the geometry follows maximum packing rules. 6.2.4 Electrical conductivity Ionic substances in solution conduct electricity because the ions are free to move and carry the electrical charge from the anode to the cathode. Ionic substances conduct electricity when molten because atoms (and thus the electrons) are mobilised. Electrons can flow directly through the ionic substance in a molten state. Example: Unspectacular behavior of common white crystalline table salt (NaCl). Salt consists of positive sodium ions (Na+) and negative chloride ions (Cl-). On the other hand the element sodium is a silvery gray metal composed of neutral atoms which react vigorously with water or air. Chlorine as an element is a neutral greenish- yellow, poisonous, diatomic gas (Cl2 ). The main principle to remember is that ions are completely different in physical and chemical properties from the neutral atoms of the element. The notation of the + and - charges on ions is very important as it conveys a definite meaning. Whereas elements are neutral in charge, IONS have either a positive or negative charge depending upon whether there is an excess of protons (positive ion) or excess of electrons (negative ion). 6.2.5 Formation of Positive Ions Metals usually have 1-4 electrons in the outer energy level. The electron arrangement of a rare gas is most easily achieved by losing the few electrons in the newly started energy level. The number of electrons lost must bring the electron number "down to" that of a prior rare gas.
Let us consider sodium, the atomic number is eleven; therefore, there are eleven electrons and eleven protons on the neutral sodium atom.
Fig 25: The Bohr diagram and Lewis symbol for sodium
This analysis shows that sodium has only one electron in its outer level. The nearest rare gas is neon with 8 electron in the outer energy level. Therefore, this electron is lost so that there are now eight electrons in the outer energy level, and the Bohr diagrams and Lewis symbols for sodium ion and neon are identical. The octet rule is satisfied. What is the charge on sodium ion as a result of losing one electron? A comparison of the atom and the ion will yield this answer. Sodium Atom Sodium Ion 11 p+ to revert to 11 p + Protons are identical in the atom and ion. 12 n an octet 12 n Positive charge is 11 elose 1 electron 10 ecaused by lac k o f 0 charge + 1 charge
Table : 1 A comparison of the atom and the ion
electrons.
An ionic compound is formed by the complete transfer of electrons from a metal to a nonmetal and the resulting ions have achieved an octet. The protons do not change. Metal atoms in Groups 1-3 lose electrons to nonmetal atoms with 5-7 electrons missing in the outer level. Non-metals gain 1-4 electrons to complete an octet. Octet rule: Elemental atoms generally lose, gain, or share electrons with other atoms in order to achieve the same electron structure as the nearest rare gas with eight electrons in the outer level. The proper application of the Octet Rule provides valuable assistance in predicting and explaining various aspects of chemical formulas.
6.2.6 Formation of Negative Ions Let us consider fluorine, the atomic number is nine, therefore, there are nine electrons and nine protons on the neutral fluorine atom.
Fig 26: The Bohr diagram and Lewis symbol for fluorine
This analysis shows that fluorine already has seven electrons in its outer level. The nearest rare gas is neon with 8 electron in the outer energy level. Therefore only one additional electron is needed to complete the octet in the fluorine atom to make the fluoride ion. If the one electron is added, the Bohr diagrams and Lewis symbols for fluorine and neon are identical. The octet rule is satisfied. What is the charge on fluorine as a result of adding one electron? A comparison of the atom and the ion will yield this answer. Fluorine Atom 9 p+ 10 n 9 eProtons are in the atom and ion. Negative charge is caused by excess 0 charge - 1 charge electrons The "ide" ending in the name signifies a simple negative ion.
Table 2 : A comparison of the fluorine atom and the fluorine ion
to complete octet add 1 electron
Fluoride Ion * 9p+ 10 n 10 e-
6.3 STABILITY OF ELECTROVALENT BOND

The stability of ionic bond can be determined by ionization energy, electron affinity, and lattice energy The ionization potential, ionization energy or EI of an atom or molecule is the energy required to remove one mole of electrons from one mole of isolated gaseous atoms or ions. More generally, the nth ionization energy is the energy required to strip it of an nth mole of electrons after the first n - 1 mole of electrons have already been removed. It is
considered in physical chemistry as a measure of the "reluctance" of an atom or ion to surrender an electron, or the "strength" by which the electron is bound; the greater the ionization energy, the more difficult it is to remove an electron. The ionization potential is an indicator of the reactivity of an element. Elements with low ionization energy tend to be reducing agents and to form salts. Factors influencing ionization energy are a) Distance of the electrons from the nucleus: The greater is the distance the smaller is the ionization energy since, the attractive force exerted by the positively charged nucleus becomes smaller the electron is remove d easily. b) Nuclear charge The greater the charge on the nucleus the more difficult it is to remove an electron. Hence, higher is the value of ionization energy c) The shielding effect of other electrons of the atom The attractive force exerted by the nucleus on the most loosely held outer electrons is partially counterbalance by the repulsive forces exerted by the inner electrons. The electro to be removed is thus shielded by the inner electron s resulting in some decrease in ionization energy 6.3.1 Electron affinity: Electro affinity depends on a) Size of the atom b) Nuclear charge The greater the nuclear charge and smaller the size, the more highly exoergic and hence most favorable for ionic bond formation The electron affinity, Eea, of an atom or molecule is the energy required to detach an electron from a singly charged negative ion, i.e., the energy change for the process X- X + eAn equivalent definition is the energy released (Einitial - E final) when an electron is attached to a neutral atom or molecule. It should be noted that the sign convention for Eea is the opposite of most thermodynamic quantities: a positive electron affinity indicates that energy is released on going from atom to anion. All elements whose EA have been measured using modern methods have a positive electron affinity, but older texts mistakenly report that some elements such as alkaline earth metals have negative Eea, meaning they would repel electrons. This is not recognized by modern chemists. The electron affinities of the noble gasses have not been conclusively measured, so they may or may not have slightly negative EAs. Atoms whose anions are relatively more stable than neutral atoms have a smaller Eea. Chlorine most strongly attracts extra electrons; mercury most weakly attracts an extra electron. Eea of noble gases are close to 0.Although Eea vary in a chaotic manner across the table, some patterns emerge. Generally, nonmetals have more positive Eea than metals.
6.3.2 Lattice energy Lattice energy deals primarily with metals. The lattice energy, or lattice enthalpy, of an ionic solid is a measure of the strength of bonds in that ionic compound. It is given the symbol U and is equivalent to the amount of energy required to separate a solid ionic compound into gaseous ions. Lattice energy can also be considered as the energy given off when gaseous ions form an ionic solid. It is dependent on ionic charge and the ionic radius: as the charge of the ions increases the lattice energy increases (becomes more negative), and as the radius decreases (the ions in the ionic solid are closer together) the lattice energy increases. It cannot be determined directly but can be by using experimental data in the Born Haber cycle and from theoretical calculations. Lattice energy is the energy change when an ionic compound is separated into its gaseous ions. It is given in units of kilojoules per mole. For example, when solid sodium chloride is separated into gaseous sodium and chlorine ions (NaCl(s) Na+(g)+Cl-(g), the lattice energy is 786 kJ/mol. Born-Mayer and Kapustinskii equations can also be used. Lattice energy is usually calculated by using the Born-Haber cycle. Lattice energy, the potential energy of two interacting ions (taken as two point charges), can also be calculated by using a modified version of Coulomb's law:
d is the distance between the centers of the ions, and q1 ,q2 are the charges on the ions.
6.4 LET US SUM UP

In this unit, we have briefly touched upon the following points Basis of chemical reactions is the forming and the breaking of bonds and the changes in bonding forces. There are two main classes of bonding forces: covalent bonds and ionic bonds. structure of ionic compounds are characterized by an orderly arrangement of its ions controlled primarily by the sizes of the ions and their charges and have no preferred directional properties, The stability of ionic bond can be determined by ionization energy, electron affinity, and lattice energy
Factors influencing ionization energy are: distance of the electrons from the nucleus, nuclear charge and the shielding effect of other electrons of the atom

Make a comparative study between covalent and ionic bonds. Analyze the highlighting points on comparison.

Examine the Octet Rule.
6.7 REFERENCES
(1) A. S. Negi, S. C. Anand - A Textbook of Physical Chemistry, 2nd edition , 2007 (2) Yadav M.S - Chemical Bonding (3) www.purchon.com/chemistry/bonding.htm (4) www.chemguide.co.uk/atoms/bonding/ionic.html (5) www.wordwebonline.com/en/ELECTROVALENTBOND
LESSON 7 - COVALENT BOND

Contents: 7.0 Aims and Objectives 7.1 Introduction 7.2 Octet Rule and covalent compounds 7.3 Coordinate covalent bond 7.4 Let us Sum Up 7.5 Lesson End Activities 7.6 Check your Progress 7.7 References

In this unit we disscuss the nature of covalent bond with its properties, and coodination covalent bond. After going through this unit, you will be able to: Decribe covalent bond and molecular orbital theory Describe octet rule Describe coordination covalent bond
7.1 INTRODUCTION
A Covalent Bond is a bond formed by the sharing of one, two or three pairs of electrons by two atoms. The bonded atoms will experience some level of increased stability as a result of the sharing process. The shared electrons will reside primarily between the two bonded nuclei. This will produce an electrostatic force of attraction between the nuclei and the shared electron cloud. This force holds the bonded system together. The more electrons occupying the area between the nuclei, the greater the attraction and the stronger the bond. Consequently, all other factors being equal, a triple bond is stronger than a double bond which is stronger than a single bond. It is not possible to ever have more than three pairs of electrons between the two bonded nuclei. Any number beyond three pairs would increase the area of negative charge so much that the extra electron pairs will always be pushed out of the region and the bond would be destroyed.
Fig 27: Covalent bond
One theory of covalent bonded is the Molecular Orbital theory. This theory states that in the covalent bonding process new orbitals, called Molecular Orbitals, are created from the original atomic orbitals that were located on the bonding atoms. In the covalent bond process, the electrons are not always shared equally. If there is a substantial difference between the electronegativities of the two bonded atoms, then the electrons will reside closer to the atom with the higher electronegativity. This will lead to a covalent bond that is polar. When the atoms have similar electronegativity values, then the electrons are shared more equally and the bond is referred to as being nonpolar.
7.2 OCTET RULE AND COVALENT COMPOUNDS

Applied to covalent bonding, the octet rules states that when atoms form covalent bonds, they tend to share sufficient electrons so as to achieve an outer shell having eight electrons (except for hydrogen and helium, which have a stable outer shell of 2 electrons). For example, here is the bonding of hydrogen Chloride (HCl):
Fig 28: Formation of hydrogen Chloride by covalent bonding
An octet is formed on the chlorine atom and the maximum pair on the hydrogen atom. Here is the bonding of a chlorine molecule (Cl2 ):
Fig 29 : Formation of Chlorine molecule by covalent bonding
An octet is formed on the two chlorine atoms. Some atoms can also form more than one covalent bond. For example, carbon can form 4 bonds (because it has 4 valence electrons), nitrogen can form 3 (5 valence electrons), and oxygen can from 2 (6 valence electrons):
Fig 30 : Covalent bonding in a)methane b)ammonia and c)water
7.3 COORDINATE COVALENT BOND

A coordinate covalent bond (also known as dative bond) is a description of covalent bonding between two atoms in which both electrons shared in the bond come from the same atom. The distinction from ordinary covalent bonding is artificial, but the terminology is popular in textbooks, especially those describing coordination compounds. Once the bonds have been formed using this, its strength and description is no different from that of other polar covalent bonds. Coordinate covalent bonds are invoked when a Lewis base (an electron donor or giver) donates a pair of electrons to a Lewis acid (an electron acceptor) to give a so-called adduct. The process of forming a dative bond is called coordination. The electron donor acquires a positive formal charge, while the electron acceptor acquires a negative formal charge. Examples Classically, any compound that contains a lone pair of electrons is capable of forming a coordinate bond. The bonding in diverse chemical compounds can be described as coordinate covalent bonding.
Fig 31: Coordinate Covalent bonding in ammonium ion
Carbon monoxide (CO) can be viewed as containing one coordinate bond and two "normal" covalent bonds between the carbon atom and the oxygen atom. This highly unusual description illustrates the flexibility of this bonding description. Thus in CO, carbon is the electron acceptor and oxygen is the electron donor. The ammonium ion (NH4 +), can be viewed as consisting of four coordinate covalent bonds between the protons (the H+ ion) and the nitrogen trianion N3 -. beryllium dichloride (BeCl2) is described as electron deficient in the sense that the triatomic species (which does exist in the gas phase) features Be centers with four valence electrons. When treated with excess chloride, the Be2+ ion binds four chloride ions to form tetrachloroberyllate anion, BeCl42-, wherein all ions achieve the octet configuration of electrons.
7.4 LET US SUM UP

A Covalent Bond is a bond formed by the sharing of one, two or three pairs of electrons by two atoms. Molecular Orbital theory states that in the covalent bonding process new orbital, called molecular Orbital A coordinate covalent bond is a description of covalent bonding between two atoms in which both electrons shared in the bond come from the same atom.

Apply octet rule to the covalent bonds and analyze the results.

Analyze the features of coordinate covalent bond (Refer 2.3).
7.7 REFERENCES
(1) Jack Barrett Structure and Bonding
(2) www.chemguide.co.uk/atoms/bonding/covalent.html (3) en.wikipedia.org/wiki/Covalent_bond
LESSON 8 - VAN DER WAALS FORCE AND METALLIC BOND

Contents 8.0 Aims and objectives 8.1 Van der Waals force 8.1.1.Introduction 8.1.2 London dispersion force 8.1.3 Lennard-Jones Potential 8.2 Metalic Bond 8.3 Let us Sum Up 8.4 Lesson End Activities 8.5 Check your Progress 8.6 Suggested Readings/References/Sources

In this unit, we have discussed about vanderwaals force and metallic bond After going through this lesson , you must be able to : Describe the properties of Vander Waals forces Describe London dispersion force and Lennard-Jones potential Describe Bonding in metals
8.1 VAN DER WAALS FORCE

The van der Waals equation is an equation of state that can be derived from a special form of the potential between a pair of molecules (hard-sphere repulsion and R-6 van der Waals attraction). In chemistry and physics, the name van der Waals force is sometimes used as a synonym for the totality of non-covalent forces (also known as intermolecular forces). These forces, which act between stable molecules, are weak compared to those appearing in chemical bonding. Historically, the use of the name for the total force is correct, because the Dutch physicist J. D. van der Waals, who lent his name to these forces, considered both the repulsive and the attractive component of the intermolecular force.Unfortunately, there is no strict convention when considering the definition of Van
der Waals force. Some texts consider only the attractive component of the intermolecular potential as the Van der Waals force. Other texts designate only a certain part of the attraction as the Van der Waals force. In general an intermolecular potential has a repulsive part, prohibiting the collapse of molecular complexes, and an attractive part. The attractive part, in turn, consists of three distinct contributions (i) The electrostatic interactions between charges (in the case of molecular ions), dipoles (in the case of molecules without inversion center), quadrupoles (all molecules with symmetry lower than cubic), and in general between permanent multipoles. The electrostatic interaction is sometimes called Keesom interaction or Keesom force after Willem Hendrik Keesom. (ii) The second source of attraction is induction (also known as polarization), which is the interaction between a permanent multipole on one molecule with an induced multipole on another. This interaction is sometimes measured in debyes after Peter J.W. Debye. (iii) The third attraction is usually named after London who himself called it dispersion. This is the only attraction experienced by noble gas atoms, but it is operative between any pair of molecules, irrespective of their symmetry. Returning to nomenclature: some texts mean by the Van der Waals force the totality of forces (including repulsion), others mean all the attractive forces (and then sometimes distinguish Van der Waals-Keesom, Van der Waals-Debye, and Van der Waals-London), and, finally some use the term "Van der Waals force" solely as a synonym for the London/dispersion force. So, if you come across the term "Van der Waals force", it is important to ascertain to which school of thought the author belongs. All intermolecular/Van der Waals forces are anisotropic (except those between two noble gas atoms), which means that they depend on the relative orientation of the molecules. The induction and dispersion interactions are always attractive, irrespective of orientation, but the electrostatic interaction changes sign upon rotation of the molecules. That is, the electrostatic force can be attractive or repulsive, depending on the mutual orientation of the molecules. When molecules are in thermal motion, as they are in the gas and liquid phase, the electrostatic force is averaged out to a large extent, because the molecules thermally rotate and thus probe both repulsive and attractive parts of the electrostatic force. Sometimes this effect is expressed by the statement that "random thermal motion around room temperature can usually overcome or disrupt them" (which refers to the electrostatic component of the Van der Waals force). Clearly, the thermal averaging effect is much less pronounced for the attractive induction and dispersion forces. The Lennard-Jones potential is often used as an approximate model for the isotropic part of a total (repulsion plus attraction) van der Waals force as a function of distance.
Van der Waals forces are responsible for certain cases of pressure broadening (van der Waals broadening) of spectral lines and the formation of van der Waals molecules. 8.1.1 London dispersion force London dispersion forces, named after the German-American physicist Fritz London, are weak intermolecular forces that arise from the attractive force between transient dipoles (or better multipoles) in molecules without permanent multipole moments. London dispersion forces are also known as dispersion forces, London forces, induced dipoleinduced dipole forces, or, as van der Waals forces. London forces can be exhibited by nonpolar molecules because electron density moves about a molecule probabilistically, see quantum mechanical theory of dispersion forces. There is a high chance that the electron density will not be evenly distributed throughout a nonpolar molecule. When an uneven distribution occurs, a temporary multipole is created. This multipole may interact with other nearby multipoles. London forces are also present in polar molecules, but they are usually only a small part of the total interaction force. Electron density in a molecule may be redistributed by proximity to another multipole. Electrons will gather on the side of a molecule that faces a positive charge and will retreat from a negative charge. Hence, a transient multipole can be produced by a nearby polar molecule, or even by a transient multipole in another nonpolar molecule. In vacuum, London forces are weaker than other intermolecular forces such as ionic interactions, hydrogen bonding, or permanent dipole-dipole interactions. This phenomenon is the only attractive intermolecular force at large distances present between neutral atoms (e.g., helium), and is the major attractive force between non-polar molecules, (e.g., nitrogen or methane). Without London forces, there would be no attractive force between noble gas atoms, and they could not then be obtained in a liquid form. London forces become stronger as the atom (or molecule) in question becomes larger. This is due to the increased polarizability of molecules with larger, more dispersed electron clouds. This trend is exemplified by the halogens (from smallest to largest: F2, Cl2, Br2, I2). Fluorine and chlorine are gases at room temperature, bromine is a liquid, and iodine is a solid. The London forces also become stronger with larger amounts of surface contact. Greater surface area means closer interaction between different molecules. The London-van der Waals forces is related to the Casimir effect for dielectric media, the former the microscopic description of the latter bulk property. The first detailed calculations of this were done in 1955 by E. M. Lifshitz.
8.1.2. Lennard-Jones Potential The Lennard-Jones potential describes the interaction between pairs of atoms in this simulation. It has the following form,
. . . . (5) where r is the distance from any atom to any other atom in the simulation. The constants (epsilon) and (sigma) are important to determine the strength and shape of the interaction and hence the properties of the solid or liquid. The force between individual atoms is of course, just the negative of the gradient of the potential. To see a graph of the potential, go to the potential panel and click the "Graph" button. Let's consider what the constants in this equation mean. Sigma describes basically where the potential equals zero. (If you plug sigma in for r you get zero.) So by changing this, you move the whole potential to the right or to the left. This can affect the overall structure of the solid state. Epsilon describes the strength of the interaction or the depth of the potential "well." In fact it can be shown that the depth of the well is just . Try changing this number in the potential panel and see what effect it has on the depth of the well. This can effect many things for the system including the freezing temperature.
8.2. METALLIC BOND

Metallic bonding is the bonding between atoms within metals. It involves the delocalized sharing of free electrons among a lattice of metal atoms. Thus, metallic bonds may be compared to molten salts.
Fig 32: Metallic bond
Metallic bonding is the electrostatic attraction between the metal atoms or ions and the delocalized electrons, also called conduction electrons. This is why atoms or layers are allowed to slide past each other, resulting in the characteristic properties of malleability and ductility. Metal atoms typically contain fewer electrons in their valence shell relative to their period or energy level. These electrons can be easily lost by the atoms and therefore become delocalized and form a sea of electrons surrounding a giant lattice of positive ions. The electrons and the positive ions in the metal have a strong attractive force between them. This means that more energy is required to negate these forces. Therefore metals often have high melting or boiling points. The principle is similar to that of ionic bonds. Metallic bonding is non-polar, because for pure elemental metals and even for alloys there is no (or a very small) electronegativity difference among the atoms participating in the bonding interaction, and the electrons involved in that interaction are delocalized across the crystalline structure of the metal. The metallic bond accounts for many physical characteristics of metals, such as strength, malleability, ductility, conduction of heat and electricity, and lustre. Due to the fact that the electrons move independently of the positive ions in a sea of negative charge, the metal gains some electrical conductivity. It allows the energy to pass quickly through the electrons generating a current. Heat conduction works on the same principle - the free electrons can transfer the energy at a faster rate than other substances such as those which are covalently bonded, as these have their electrons fixed into position. There also are few nonmetals which conduct electricity: graphite (because, like metals, they have free electrons), and molten and aqueous ionic compounds which have free moving ions. Metal atoms do not have at least one valence electron which they do not share with neighboring atoms, nor do they lose electrons to form ions. Instead the outer energy levels of the metal atoms overlap. They are similar to covalent bonds.Metallic bonding was first discovered by Frederick Louise in 1922. There are many theories available to explain metallic bond 1. Free electron theory : 2. Valence bond theory, Paulings theory or resonance theory 3. Band or Zone Theory
8.3 LET US SUM UP

The van der Waals force represents the electromagnetic interaction of fluctuating dipoles in the atoms of the tip and surface. The Lennard-Jones potential is often used as an approximate model for the isotropic part of a total (repulsion plus attraction) van der Waals force as a function of distance. Van der Waals forces are responsible for certain cases of pressure broadening (van der Waals broadening) of spectral lines and the formation of van der Waals molecules. Metallic bonds involves the delocalized sharing of free electrons among a lattice of metal atoms

Out of the theories explaining the metallic bond, which are is very clear in explaining it? Give valid reasons for the same.

Is metallic bonding polar or non-polar? Give your reasons.
8.6 REFERENCES
en.wikipedia.org/wiki/Metallic_bond www.chemguide.co.uk/atoms/bonding/metallic.html www.tpub.com/content/doe/h1015v1/css/h1015v1_49.htm
LESSON 9 - MOLECULAR GEOMETRY AND SHAPE OF ORBITALS

Contents 9.0 Aims and objectives 9.1 Molecular Geometry 9.1.1 Introduction 9.1.2 The influence of thermal excitation 9.1.3 Bonding 9.1.4 Isomers 9.2 Shapes of Atomic Orbitals 9.2.1 s Orbitals 9.2.2 p Orbitals 9.2.3 d Orbitals 9.2.4 f Orbitals 9.3 Types of Molecular Structure 9.4 Let us Sum Up 9.5 Suggested Readings/References/Sources

This section will deal with the molecular geometry and shape of atomic orbitals After reading this unit, you must be able to Describe the geometry of various molecules and bonding Describe and draw the shapes of different orbitals Describe the types of molecular structure
9.1 MOLECULAR GEOMETRY

9.1.1. Introduction
Molecular geometry or molecular structure is the three-dimensional arrangement of the atoms that constitute a molecule, inferred from the spectroscopic studies of the compound. It determines several properties of a substance including its reactivity, polarity, phase of matter, color, magnetism, and biological activity. Molecular geometries are best determined at temperatures close to absolute zero because at higher temperatures the molecules will show considerable rotational motion. In the solid state the molecular geometry can be measured by X-ray crystallography. Geometries can be computed by quantum mechanical calculations or by semi-empirical molecular modeling. Larger molecules often exist in multiple stable chemical conformations that differ in their molecular geometry and are separated by high hills in the potential energy surface. The position of each atom is determined by the nature of the chemical bonds by which it is connected to its neighboring atoms. The molecular geometry can be described by the positions of these atoms in space, evoking bond lengths of two joined atoms, bond angles of three connected atoms, and torsion angles of three consecutive bonds.
Fig 33: Geometry of the water molecule
9.1.2 The influence of thermal excitation Since the motions of the atoms in a molecule are determined by quantum mechanics, one must define "motion" in a quantum mechanical way. The overall (external) quantum mechanical motions translation and rotation hardly change the geometry of the molecule. (To some extent rotation influences the geometry via Coriolis forces and centrifugal distortion, but this is negligible for the present discussion.) A third type of motion is vibration, which is the internal motion of the atoms in a molecule. The molecular vibrations are harmonic (at least to good approximation), which means that the atoms oscillate about their equilibrium, even at the absolute zero of temperature. At absolute zero all atoms are in their vibrational ground state and show zero point quantum
mechanical motion, that is, the wavefunction of a single vibrational mode is not a sharp peak, but an exponential of finite width. At higher temperatures the vibrational modes may be thermally excited (in a classical interpretation one expresses this by stating that "the molecules will vibrate faster"), but they oscillate still around the recognizable geometry of the molecule. To get a feeling for the probability that the vibration of molecule may be thermally excited, we inspect the Boltzmann factor ,
where E is the excitation energy of the vibrational mode, k the Boltzmann constant and T the absolute temperature. At 298K (25 0C), typical values for the Boltzmann factor are: E = 500 cm-1 --> 0.089; E = 1000 cm-1 --> 0.008; E = 1500 cm-1 --> 7 10-4 . That is, if the excitation energy is 500 cm-1 , about 9% of the molecules are thermally excited at room temperature. The lowest excitation vibrational energy in water is the bending mode (about 1600 cm-1 ). Thus, at room temperature less than 0.07% of all the molecules of a given amount of water will vibrate faster than at absolute zero. As stated above, rotation hardly influences the molecular geometry. But, as a quantum mechanical motion, it is thermally excited at relatively (as compared to vibration) low temperatures. From a classical point of view it can be stated that more molecules rotate faster at higher temperatures, i.e., they have larger angular velocity and angular momentum. In quantum mechanically language: more eigenstates of higher angular momentum become thermally populated with rising temperatures. Typical rotational excitation energies are on the order of a few cm-1 . The results of many spectroscopic experiments are broadened because they involve an averaging over rotational states. It is often difficult to extract geometries from spectra at high temperatures, because the number of rotational states probed in the experimental averaging increases with increasing temperature. Thus, many spectroscopic observations can only be expected to yield reliable molecular geometries at temperatures close t o absolute zero, because at higher temperatures too many higher rotational states are thermally populated. 9.1.3 Bonding Molecules, by definition, are most often held together with covalent bonds involving single, double, and/or triple bonds, where a "bond" is a shared pair of electrons (the other method of bonding between atoms is called ionic bonding and involves a positive cation and a negative anion). Molecular geometries can be specified in terms of bond lengths, bond angles and torsional angles. The bond length is defined to be the average distance between the centers of two atoms bonded together in any given molecule. A bond angle is the angle
formed by three atoms bonded together. For four atoms bonded together in a straight chain, the torsional angle is the angle between the plane formed by the first three atoms and the plane formed by the last three atoms. Molecular geometry is determined by the quantum mechanical behaviour of the electrons. Using the valence bond approximation this can be understood by the type of bonds between the atoms that make up the molecule. Before atoms interact to form a chemical bond, the atomic orbitals mix in a process called orbital hybridisation.The two most common types of bonds are Sigma bonds and Pi bonds. The geometry can also be understood by molecular orbital theory where the electrons are delocalised. An understanding of the wavelike behavior of electrons in atoms and molecules is the subject of quantum chemistry. 9.1.4 Isomers Isomers are types of molecules that share a chemical formula but have different geometries, resulting in very different properties: A pure substance is composed of only one type of isomer of a molecule (all have the same geometrical structure). Structural isomers have the same chemical formula but different physical arrangements, often forming alternate molecular geometries with very different properties. The atoms are not bonded (connected) together in the same orders. Functional isomers are special kinds of structural isomers, where certain groups of atoms exhibit a special kind of behavior, such as an ether or an alcohol. Stereoisomers may have many similar physicochemical properties (melting point, boiling point) and at the same time very different biochemical activities. This is because they exhibit a handedness that is commonly found in living systems. One manifestation of this chirality or handedness is that they have the ability to rotate polarized light in different directions. Protein folding concerns the complex geometries and different isomers that proteins can take.
9.2 SHAPE OF ATOMIC ORBITALS

The quantum- mechanical treatment of electrons in atoms gives a clear picture of the energy levels associated with every atomic orbital which can be defined by a set of four quantum numbers. The question of where in space the electron may be is a somewhat more ambiguous question and receives a somewhat more ambiguous answer. Solutions of the Schrdinger equation correspond to waves rather than to particles, and at least in principle all electromagnetic waves extend throughout all space! This interesting result is not helpful in locating the physical electron on a given atom.
The location of an electron can be described in either of two equivalent ways using the quantum- mechanical results. If the electron is visualized as a real very small object moving very rapidly, then the space it occupies can be described in terms of the probability of finding the electron at a given point or within a given space at any instant. If the desired probability is set at 99%, or 95%, a physical space occupied by the electron can be calculated on this time-average basis. If, on the other hand, the electron is visualized as an electromagnetic wave, then the amplitude of that wave, or the wave function, will be greater at some locations than at others. The space the electron occupies can be considered to be the space within which the amplitude of its wave function is greater than 1%, or 5%, of its maximum amplitude. Again, a three-dimensional space "containing" the electron will be defined. The electron can be described equally well in either way or the three-dimensional spaces defined as "containing" the electron as a wave and as a particle are the same. Chemists find it convenient to describe the location of electrons in atoms and molecules in terms of this type of shape. These representations of orbital shapes are those which are said to contain 99% or 95% of the electron density of the orbital. In a previous section, the shapes of the orbitals were assumed to be spherical and atomic and ionic radii could be calculated on that basis. In the quantum- mechanical treatment of atoms, ions, and molecules, many of the orbitals are not found to be spherical in shape. Moreover, the different orbitals on the same atom do interpenetrate each other and the electronic structure of the outer atom is to some extent a composite of several orbitals. For these reasons the atomic radius and ionic radius are now viewed as useful empirical measurements of the sizes of atoms and ions rather than as properties with fundamental significance. Even so, practicing chemists still learn much about the structures of compounds using molecular models made up of scale models of spherical atoms. The values of all four quantum numbers influence the location of an electron, or in the terminology just introduced the distribution of electron density in space or the shape of an orbital, but the effects of the four different numbers are not the same. The principal quantum number n affects primarily the size of the orbital and has a lesser influence on its shape. The subshell quantum number l affects primarily the shape of the orbital. The magnetic quantum number m affects primarily the orientation of the orbital in threedimensional space. The spin quantum number s has little effect upon the location of the orbitals of an isolated atom, but does have an influence on orbital interactions when the orbitals of different atoms impinge upon each other.
a.
b.
Fig 34 : Shape of s orbital and p orbital
Electron density relates to how much of an electron's charge is packed into a given volume. In dense places on the dot-diagram, there is a high concentration of electrical charge.
a. 9.2.1 s Orbitals
b.
c.
Fig 35 : Shape of a)px orbital b)py orbital c)pz orbital
Orbitals with subshell quantum number l = 0 are called s orbitals. All s orbitals are spherical in shape and have spherical symmetry. This means that the wave function will depend only on the distance from the nucleus and not on the direction. In any atom, the size of the s orbital increases as the principal quantum number of the orbital increases but the geometry remains spherical. The electron density also tends to extend further. Other orbitals behave in the same way as the principal quantum numbers of the orbitals increase. 9.2.2 p Orbitals Orbitals with subshell quantum number l = 1 are called p orbitals. Since the magnetic quantum number m can be -1, 0, or +1 when the value of the subshell quantum number l is one, p orbitals come in sets of three. In each set, one of the orbitals is aligned along each of the three mutually perpendicular axes of the atom; these axes are traditionally designated x, y, and z. The three 2p orbitals are correspondingly designated 2px , 2py , and 2pz. The p orbitals either as a set or individually do not have spherical symmetry and so a
simple plot of radial probability density cannot be made for them. If, however, the distance from the nucleus is taken along any one of the three axes and the orbital is that along the same axis, then a suitable plot can be made. 9.2.3 d Orbitals Orbitals with subshell quantum number l = 2 are called d orbitals; since m can be -2, -1, 0, +1, or +2 when l is two, d orbitals come in sets of five. The d orbitals, and the more complex f orbitals, are usually visualized in three-dimensional representations, even if these have to be shown on a two-dimensional page. 9.2.4 f Orbitals Orbitals with subshell quantum number l = 3 are called f orbitals. These orbitals are found only in the lanthanide and actinide elements. Since m can be -3, -2, -1, 0, +1, +2, or +3 when l has the value 3, f orbitals come in sets of seven. The f orbitals are rarely of direct chemical interest because they tend to be buried deep within the electronic cloud of an atom, but they do play a major role in the spectroscopy of the lanthanides and actinides. The f orbitals are the most complex orbitals with which most chemists are concerned, even though 5g orbitals, with quantum number l = 4, are known to exist.
a.
b.
c.
d.
e.
Fig 36: shape of a)2dxy orbital b) dxz orbital c) dyz orbital d) dx2 -y2 orbital e) dz2 orbital
9.3 TYPES OF MOLECULAR STRUCTURE

There are Six basic shape types for molecules A) Linear In a linear model, atoms are connected in a straight line. The bond angles are set at 180.A bond angle is very simply the geometric angle between two adjacent bonds. B)Trigonal Planar
Just from its name, it can easily be said that molecules with the trigonal planar shape are somewhat triangular and in one plane (meaning a flat surface). Consequently, the bond angles are set at 120. An example of this is boron trifluoride. CTetrahedral Tetra- signifies four, and - hedral relates to a surface, so tetrahedral almost literally means "four surfaces." This is when there are four bonds all on one central atom, with no extra unshared electron pairs. In accordance with the VSEPR (valence-shell electron pair repulsion theory), the bond angles between the electron bonds are 109.5. An example of a tetrahedral molecule is methane (CH4 ). D)Octahedral Octa- signifies eight, and - hedral relates to a surface, so octahedral almost literally means "eight surfaces." E)Pyramidal Pyramidal-shaped molecules have pyramid- like shapes. Unlike the linear and trigonal planar shapes but similar to the tetrahedral orientation, pyramidal shapes requires three dimensions in order to fully separate the electrons. Here, there are only three pairs of bonded electrons, leaving one unshared pair. The bond angles are 107. An example is NH3 (ammonia). F)Bent The final basic shape of a molecule is the bent shape. One of the most unquestionably important molecules any chemist studies is water, or H2 O. A water molecule has a bent shape because it has two pairs of bonded electrons and two unshared pairs. Like in the other arrangements, electrons must be spaced as far as possible. Therefore, the bond angles here are 104.5.
Table 3 : Basic shape types for molecules
9.4 LET US SUM UP

Geometries can be computed by quantum mechanical calculations or by semiempirical molecular modeling The position of each atom is determined by the nature of the chemical bonds by which it is connected to its neighboring atoms Molecular geometry is determined by the quantum mechanical behaviour of the electrons A pure substance is composed of only one type of isomer of a molecule. There are Six basic shape types for molecules

1. Elaborate on molecular geomentry of the orbitals.

Make a comparative study on s, p, d and f obitals.
9.5 REFERENCES
(1) John C. Kotz, Paul Treichel, (2) Gabriela C. Weaver (3) D. Nasipuri Stereochemistry of Organic Compounds Chemistry & Chemical Reactivity
(4) Daintith, J. (2004).
Oxford Dictionary of Chemistry. New York: Oxford University Press
(5) http://intro.chem.okstate.edu/1314F00/Lecture/Chapter10/VSEPR.html
LESSON 10 - HYBRIDIZATION OF ATOMIC ORBITALS AND VSEPR THEORY

Contents 10.0 Aims and objectives 10.1 Hybridization of Atomic Orbitals 10.1.1 Introduction 10.1.2 Hybridization in few molecules 10.1.3 Hybridization Involving d-Orbitals 10.1.4 Hybridization Involving Multiple Bonds 10.2 VSEPR theory 10.3 Let us Sum Up 10.4 Lesson End Activities 10.5 Check your Progress 10.6 References

This section will deal with the hybridization of atomic orbital with few examples. We also discussed the use of VSEPR theory for the representation of shapes of individual molecules, based upon their extent of electron-pair electrostatic repulsion, determined using steric numbers. After reading this unit, you should be able to Describe hybridization in various molecules To know the use of VSEPR theory for the representation of individual molecules
10.1 HYBRIDIZATION OF ATOMIC ORBITALS

10.1.1 Introduction If the four hydrogen atoms in a methane molecule (CH4 ) were bound to the three 2p orbitals and the 2s orbital of the carbon atom, the H-C-H bond angles would be 90o for 3 of the hydrogen atoms and the 4th hydrogen atom would be at 135o from the others. Experimental evidence has shown that the bond angles in methane are not arranged that way but are 109.5o giving the overall shape of a tetrahedron. The tetrahedral structure makes much more sense in that hydrogen atoms would naturally repel each other due to
their negative electron clouds and form this shape. If you think electron-electron repulsion isn't significant, try walking through a wall! There is plenty of space for your nuclei to pass through the nuclei of the wall material but ouch, it just doesn't work that way. Experimental evidence has also shown that the H-N-H bond angles in ammonia (NH3 ) are 107o and the H-O-H bond angles in water are 105o . It is clear from these bond angles that the non-bonding pairs of electrons occupy a reasonable amount of space and are pushing the hydrogen atoms closer together compared to the angles found in methane. The valence shell electron-pair repulsion model (VESPR) was devised to account for these molecular shapes. In this model, atoms and pairs of electrons will be arranged to minimize the repulsion of these atoms and pairs of electrons. Since the non-bonded electron pairs are held somewhat closer to the nucleus than the attached hydrogen atoms, they tend to crowd the hydrogen atoms. Thus ammonia exists as a distorted tetrahedron (trigonal pyramidal) rather than a trigonal plane and water also exists as a distorted tetrahedron (bent) rather than a linear molecule with the hydrogen atoms at a 180o bond angle. This concept proposes that since the attached groups are not at the angles of the p orbitals and their atomic orbitals would not have maximum overlap (to form strong bonds) the s and p orbitals will be hybridized to match the bond angles of the attached groups. The number of these new hybrid orbitals must be equal to the numbers of atoms and nonbonded electron pairs surrounding the central atom. 10.1.2 Hybridization in few molecules In the case of methane, the three 2p orbitals of the carbon atom are combined with its 2s orbital to form four new orbitals called "sp3 " hybrid orbitals. The name is simply a tally of all the orbitals that were blended together to form these new hybrid orbitals. Four hybrid orbitals were required since there are four atoms attached to the central carbon atom. These new orbitals will have energy slightly above the 2s orbital and below the 2p orbitals as shown in the following illustration. Notice that no change occurred with the 1s orbital.
Fig 37: Hybridization in methane
These hybrid orbitals have 75% p-character and 25% s-character which gives them a shape that is shorter and fatter than a p-orbital. The new shape looks a little like.
Fig 38 : Shape of orbitals in methane
A stick and wedge drawing of methane shows the tetrahedral angles... (The wedge is coming out of the paper and the dashed line is going behind the paper. The solid lines are in the plane of the paper.)
Fig 39 : A stick and wedge drawing of methane
In the case of ammonia, the three 2p orbitals of the nitrogen atom are combined with the 2s orbital to form four sp3 hybrid orbitals. The non-bonded electron pair will occupy a hybrid orbital. Again we need a hybrid orbital for each atom and pair of non-bonding electrons. Ammonia has three hydrogen atoms and one non-bonded pair of electrons when we draw the electron-dot formula. In order to determine the hybridization of an atom, you must first draw the electron-dot formula.
A stick and wedge drawing of ammonia showing the non-bonding electrons in a probability area for the hybrid orbital.
Fig 40:A stick and wedge drawing of ammonia
In the case of water, the three 2p orbitals of the oxygen atom are combined with the 2s orbital to form four sp3 hybrid orbitals. The two non-bonded electron pairs will occupy hybrid orbitals. Again we need a hybrid orbital for each atom and each pair of nonbonding electrons. Water has two hydrogen atoms and two non-bonded pairs of electrons when we draw the electron-dot formula.
Fig 41 : Hybridization in water.
A stick and wedge drawing of water showing the non-bonding electron pairs in probability areas for the hybrid orbital...
Fig 42: A stick and wedge drawing of water.
Now let's look at something a bit different. In the boron trifluoride molecule, only three groups are arranged around the central boron atom. In this case, the 2s orbital is combined with only two of the 2p orbitals (since we only need three hybrid orbitals for the three groups...thinking of groups as atoms and non-bonding pairs) forming three hybrid orbitals called sp2 hybrid orbitals. The other p-orbital remains unhybridized and is at right angles to the trigonal planar arrangement of the hybrid orbitals. The trigonal planar arrangement has bond angles of 120o .
Fig 43: Hybridization in boron trifluoride molecule.
In the following stick model, the empty p orbital is shown as the probability area...one end shaded blue and the other is white...there are no electrons in this orbital!
Fig 44 : A stick and wedge drawing in boron trifluoride molecule.
Finally let's look at beryllium dichloride. Since only two groups are attached to beryllium, we only will have two hybrid orbitals. In this case, the 2s orbital is combined with only one of the 2p orbitals to yield two sp hybrid orbitals. The two hybrid orbitals will be arranged as far apart as possible from each other with the result being a linear arrangement. The two unhybridized p-orbitals stay in their respective positions (at right angles to each other) and perpendicular to the linear molecule.
Fig 45 : Hybridization in beryllium dichloride .
In the following stick model, the empty p orbitals are shown as the probability areas...one green and one blue.
Fig 46 : A stick and wedge drawing of beryllium dichloride.
10.1.3 Hybridization Involving d-Orbitals As we discussed earlier, some 3rd row and larger elements can accommodate more than eight electrons around the central atom. These atoms will also be hybridized and have very specific arrangements of the attached groups in space. The two types of hybridization involved with d orbitals are sp3 d and sp3 d2 . The groups will be arranged in a trigonal bipyramidal arrangement with sp3 d hybridization...bond angles will be 120o in the plane with two groups arranged vertically above and below this plane.
There will be an octahedral arrangement with sp3 d2 hybridization...all bond angles are at 90o .
Non-bonded electron pairs are always placed where they will have the most space...in the trigonal plane for sp3 d hybridization. 10.1.4 Hybridization Involving Multiple Bonds Only a maximum of two electrons can occupy any orbital whether it is an atomic orbital or a molecular orbital due to electron-electron repulsion. When we draw a double or a triple-bond between two atoms, we imply that either four or six electrons are directly between these two atoms. Since this is impossible, we must have these extra electrons off to the side in what we refer to as pi bonds. Therefore, all multiple bonds are composed of two different kinds of molecular bonds called pi-bonds and sigma-bonds. The sigma-bond is defined as the linear overlap of atomic orbitals (hybrids except for hydrogen) in which two electrons are directly between the two bonded nuclei. Pi-bonds are defined as the parallel overlap of p-orbitals. A double bond has one sigmabond and one pi-bond. A triple bond thus consists of a sigma-bond both carbon atoms and two pi-bonds with the pi-bonds in different planes. In the molecule C2 H4 , ethene, will be sp2 hybridized and have one unpaired electron in a non-hybridized p orbital.
Fig 47 : Hybridization in ethene .
These p-orbitals will undergo parallel overlap and form one pi bond with bean-shaped probability areas above and below the plane of the six atoms. This pair of bean-shaped probability areas constitutes one pi-bond and the pair of electrons in this bond can be found in either bean-shaped area.
Fig 48 : The 3-dimensional model of ethene
The 3-dimensional model of ethene is therefore planar with H-C-H and H-C-C bond angles of 120o ...the pi-bond is not shown in this picture. Now let's look at H2 C2 (acetylene). Both carbon atoms will be sp hybridized and have one electron in each of two unhybridized p orbitals.
Fig 49 : Hybridization in acetylene.
These p orbitals will undergo parallel overlap to form two pi-bonds at right angles to each other.
Fig 50: The 3-dimensional model of acetylene
The 3-dimensional model of acetylene is therefore linear...the pi-bonds are not shown in this picture.
10.2 VSEPR THEORY

Valence shell electron pair repulsion (VSEPR) theory (1957) is a model in chemistry, which is used for the representation of shapes of individual molecules, based upon their extent of electron-pair electrostatic repulsion, determined using steric numbers. T h e
theory is also called the Gillespie-Nyholm theory after the two main developers. The premise of VSEPR is that a constructed Lewis structure is expanded to show all lone pairs of electrons alongside protruding and projecting bonds, for predicting the geometric shape and lone-pair behavior of a compound through consideration of the total coordination number. VSEPR theory is based on the idea that the geometry of a molecule or polyatomic ion is determined primarily by repulsion among the pairs of electrons associated with a central atom. The pairs of electrons may be bonding or nonbonding (also called lone pairs). Only valence electrons of the central atom influence the molecular shape in a meaningful way.
The basic assumptions of this theory are a) Pairs of electrons in the valence shell of a central atom repel each other. b) These pairs of electrons tend to occupy positions in space that minimize repulsions and maximize the distance of separation between them. c) The valence shell is taken as a sphere with electron pairs localizing on the spherical surface at maximum distance from one another. d) A multiple bond is treated as if it is a single electron pair and the two or three electron pairs of a multiple bond are treated as a single super pair. e) Where two or more resonance structures can depict a molecule the VSEPR model is applicable to any such structure. Three types of repulsion take place between the electrons of a molecule: v The lone pair- lone pair repulsion v The lone pair-bonding pair repulsion v The bonding pair-bonding pair repulsion. A molecule must avoid these repulsions to remain stable. When repulsion cannot be avoided, the weaker repulsion (i.e. the one that causes the smallest deviation from the ideal shape) is preferred. The lone pair- lone pair (lp-lp) repulsion is considered to be stronger than the lone pairbonding pair (lp-bp) repulsion, which in turn is stronger than the bonding pair-bonding pair (bp-bp) repulsion. Hence, the weaker bp-bp repulsion is preferred over the lp-lp or lp-bp repulsion. Larger molecules which fail to even maintain 90 between their electron pairs prefer to lie in more than one plane. VSEPR theory is usually compared (but not part of) and contrasted with valence bond theory, which addresses molecular shape through orbitals that are energetically accessible for bonding. Valence bond theory concerns itself with the formation of sigma and pi bonds. Molecular orbital theory is a more sophisticated model for understanding how atoms and electrons are assembled into molecules and polyatomic ions.
VSEPR theory has long been criticized for not being quantitative, and therefore limited to the generation of "crude", even though structurally accurate, molecular geometries of covalent molecules. STR3DI32 is a molecular mechanics program that quantitatively implements all of the features of VSEPR theory, and provides highly accurate simulations of molecular geometries and stereo-electronic effects. It can handle large molecules that cannot be handled by the molecular orbital methods and guest/host molecular complexes and solvation models. The AXE method is commonly used in formatting molecules to fit the VSEPR model, though the letter "B" sometimes replaces the "X".
10.3 LET US SUM UP

Hybridization of Atomic Orbitals offers a model for understanding bonding in molecules. Non-bonded electron pairs are always placed where they will have the most space...in the trigonal plane for sp3 d hybridization. The sigma-bond is defined as the linear overlap of atomic orbitals (hybrids except for hydrogen) in which two electrons are directly between the two bonded nuclei. Pi-bonds are defined as the parallel overlap of p-orbitals. Pairs of electrons in the valence shell of a central atom repel each other. Where two or more resonance structures can depict a molecule the VSEPR model is applicable to any such structure.

Hybradization of atomic orbitals offers a model for understanding bonding in moleculars Comment on it.

Give a qualitative analysis of VSEPR theory (Refer 5.2).
10.6 REFERENCES
(1) Herbert Meislich, Jacob Sharefkin - Organic Chemistry
(2) John C. Kotz, Paul Treichel, Gabriela C. Weaver- Chemistry & Chemical Reactivity (3) Stuart Rosenfeld (4) Gail Blasser Riley, Barker, Brett Barker, Arco - Basic Skills for Organic Chemistry - Chemistry
LESSON 11- PROTEIN FOLDING

Contents: 11.0 Aims and Objectives 11.1 Introduction 11.2 Known Facts about the Protein Folding Process 11.2.1 The Relationship between Folding and Amino Acid Sequence 11.2.2 Kinetics and the Levinthal Paradox 11.2.3 Techniques for Studying Protein Folding 11.2.4 Modern Studies of Folding with High Time Resolution 11.2.5 Energy Landscape Theory of Protein Folding 11.2.6 Computational Prediction of Protein Tertiary Structure 11.2.7 Techniques for Determination of Protein Structure 11.3 Let us sum up 11.4 Lesson End Activities 11.5 Check your Progress 11.6 Suggested Readings/References/Sources

This section deals with the protein folding and techniques used for the determination of protein folding. After going through this lesson, you must be able to: To Known Facts about the Protein Folding Process List various techniques for studying protein folding To describe the computational methods for prediction of 30 stucture of proteins
11.1 INTRODUCTION
Protein folding is the physical process by which a polypeptide folds into its characteristic three-dimensional structure. Each protein begins as a polypeptide, translated from a sequence of mRNA as a linear chain of amino acids. This polypeptide lacks any developed three-dimensional structure However each
amino acid in the chain can be thought of having certain 'gross' chemical features. These may be hydrophobic, hydrophilic, or electrically charged, for example. These interact with each other and their surroundings in the cell to produce a well-defined, three dimensional shape, the folded protein is known as the native state. The resulting three-dimensional structure is determined by the sequence of the amino acids. The mechanism of protein folding is not completely understood. Experimentally determining the three dimensional structure of a protein is often very difficult and expensive. However the sequence of that protein is often known. Therefore scientists have tried to use different biophysical techniques to manually fold a protein. That is, to predict the structure of the complete protein from the sequence of the protein. For many proteins the correct three dimensional structures is essential to function. Failure to fold into the intended shape usually produces inactive proteins with different properties. Several neurodegenerative and other diseases are believed to result from the accumulation of misfolded (incorrectly folded) proteins.
11.2 KNOWN FACTS ABOUT THE PROTEIN FOLDING PROCESS

11.2.1 The Relationship between Folding and Amino Acid Sequence The amino-acid sequence (or primary structure) of a protein predisposes it towards its native conformation or conformations. It will fold spontaneously during or after synthesis. While these macromolecules may be regarded as "folding themselves", the mechanism depends equally on the characteristics of the cytosol, including the nature of the primary solvent (water or lipid), the concentration of salts, the temperature, and molecular chaperones. Most folded proteins have a hydrophobic core in which side chain packing stabilizes the folded state, and charged or polar side chains on the solvent-exposed surface where they interact with surrounding water molecules. It is generally accepted that minimizing the number of hydrophobic sidechains exposed to water is the principal driving force behind the folding process, although a recent theory has been proposed which reassesses the contributions made by hydrogen bonding. The process of folding in vivo often begins co-translationally, so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome. Specialized proteins called chaperones assist in the folding of other proteins. A well studied example is the bacterial GroEL system, which assists in the folding of globular proteins. In eukaryotic organisms chaperones are known as heat shock proteins. Although most globular proteins are able to assume their native state unassisted, chaperone-assisted folding is often necessary in the crowded intracellular environment to prevent aggregation; chaperones are also used to prevent misfolding and aggregation which may occur as a consequence of exposure to heat or other changes in the cellular environment. For the most part, scientists have been able to study many identical molecules folding together en masse. At the coarsest level, it appears that in transitioning to the native state, a given amino
acid sequence takes on roughly the same route and proceeds through roughly the same intermediates and transition states. Often folding involves first the establishment of regular secondary and supersecondary structures, particularly alpha helices and beta sheets, and afterwards tertiary structure. Formation of quaternary structure usually involves the "assembly" or "coassembly" of subunits that have already folded. The regular alpha helix and beta sheet structures fold rapidly because they are stabilized by intramolecular hydrogen bonds, as was first characterized by Linus Pauling. Protein folding may involve covalent bonding in the form of disulfide bridges formed between two cysteine residues or the formation of metal clusters. Shortly before settling into their more energetically favourable native conformation, molecules may pass through an intermediate "molten globule" state. The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that identical amino acid sequences always fold similarly. Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found. Folding is a spontaneous process independent of energy inputs from nucleoside triphosphates. The passage of the folded state is mainly guided by hydrophobic interactions, formation of intramolecular hydrogen bonds, and van der Waals forces, and it is opposed by conformational entropy, which must be overcome by extrinsic factors such as chaperones. In certain solutions and under some conditions proteins will not fold into their biochemically functional forms. Temperatures above (and sometimes those below) the range that cells tend to live in will cause proteins to unfold or "denature" (this is why boiling makes the white of an egg opaque). High concentrations of solutes, extremes of pH, mechanical forces, and the presence of chemical denaturants can do the same. A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called random coil. Under certain conditions some proteins can refold; however, in many cases denaturation is irreversible.Cells sometimes protect their proteins against the denaturing influence of heat with enzymes known as chaperones or heat shock proteins, which assist other proteins both in folding and in remaining folded. Some proteins never fold in cells at all except with the assistance of chaperone molecules, which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, giving them a second chance to refold properly. This function is crucial to prevent the risk of precipitation into insoluble amorphous aggregates. 11.2.2 Kinetics and the Levinthal Paradox The entire duration of the folding process varies dramatically depending on the protein of interest. The slowest folding proteins require many minutes or hours to fold, primarily due to proline isomerizations or wrong disulfide bond formations, and must pass through the other hand, very small single-domain proteins with lengths of up to a hundred amino acids typically fold in a single step. Time scales of milliseconds are the norm and the very fastest known protein folding reactions are complete within a few microseconds.
The Levinthal paradox observes that if a protein were to fold by sequentially sampling all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the nanosecond or picosecond scale). Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur in folding, and the protein must, therefore, fold by a directed process.
11.3 TECHNIQUES FOR STUDYING PROTEIN FOLDING

11.3.1 Modern studies of folding with high time resolution The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. These are experimental methods for rapidly triggering the folding of a sample of unfolded protein, and then observing the resulting dynamics. Fast techniques in widespread use include ultrafast mixing of solutions, photochemical methods, and laser temperature jump spectroscopy. Among the many scientists who have contributed to the development of these techniques are Heinrich Roder, Harry Gray, Martin Gruebele, Brian Dyer, William Eaton, Sheena Radford, Chris Dobson, Sir Alan R. Fersht and Bengt Nlting. 11.3.2 Energy landscape theory of protein folding The protein folding phenomenon was largely an experimental endeavor until the formulation of energy landscape theory by Joseph Bryngelson and Peter Wolynes in the late 1980s and early 1990s. This approach introduced the principle of minimal frustration, which asserts that evolution has selected the amino acid sequences of natural proteins so that interactions between side chains largely favor the molecule's acquisition of the folded state. Interactions that do not favor folding are selected against, although some residual frustration is expected to exist. A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (coined by Jos Onuchic) that are largely directed towards the native state. This "folding funnel" landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by computational simulations of model proteins and has been used to improve methods for protein structure prediction and design. 11.3.3 Computational prediction of protein tertiary structure De novo or ab initio techniques for computational protein structure prediction are related to, but strictly distinct from, studies involving protein folding. Molecular Dynamics (MD) is an important tool for studying protein folding and dynamics in silico. Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and very small proteins. MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. In order to simulate long time folding processes (beyond about 1 microsecond), like folding of small-size proteins (about 50 residues) or larger, some approximations or simplifications in protein models need to be introduced. An approach using reduced protein representation (pseudo-atoms representing groups of atoms are
defined) and statistical potential is not only useful in protein structure prediction, but is also capable of reproducing the folding pathways.
11.3.4 Techniques for determination of protein structure The determination of the folded structure of a protein is a lengthy and complicated process, involving methods like X-ray crystallography and NMR. One of the major areas of interest is the prediction of native structure from amino-acid sequences alone using bioinformatics and computational simulation methods.
11.4 LET US SUM UP

Protein folding is the physical process by which a polypeptide folds into its characteristic three-dimensional structure. The macromolecules may be regarded as "folding themselves", the mechanism depends equally on the characteristics of the cytosol, including the nature of the primary solvent \ (water or lipid), the concentration of salts, the temperature, and molecular chaperones. The entire duration of the folding process varies dramatically depending on the protein of interest. Molecular Dynamics (MD) is an important tool for studying protein folding and dynamics in silico.

Explain and comment on the facts on protein folding process.

Analyze the statement that Protein folding is just a physical process (Refer 1.1).
1.7 SUGGESTED READINGS/REFERENCES/SOURCES

1. Bret A. Shirley 2. Arthur Horwich 3. R. A. Broglia, Eugene I. Shakhnovich Guido Tiana - Protein Stability and Folding - Protein Folding in the Cell
- Protein Folding, Evolution and Design
4. http://en.wikipedia.org/wiki/Protein_folding http://folding.stanford.edu/
Lesson 12- PROTEIN DENATURATION Contents: 12.0 Aims and Objectives 12.1 Introduction 12.2 Measuring Protein Denaturation 12.2.1 Loss of Solubility 12.2.2 Increased Proteolysis 12.2.3 Loss of Biological Activity 12.2.4 Tritium- Hydrogen Exchange 12.2.5 Spectroscopic Procedures 12.3 Causes of Protein Denaturation 12.3.1 Thermal Denaturation 12.3.2 pH Denaturation 12.3.3 Changes in Dielectric Constant 12.3.4 Denaturation at Interfaces 12.3.5 Ionic Strength 12.3.6 The Effect of Protein Cross linkers 12.4 Diseases Related to Protein Folding 12.4.1 Familial Amyloidotic 12.4.2 Alzheimer s disease 12.4.3 Mad Cow and Other Species 12.5 Treating Protein Misfolding 12.6 Let us sum up 12.7 Lesson End Activities 12.8 Check your Progress 12.9References

In this unit, we have discussed the problem of measuring protein denaturation, various causes for denaturation, diseases related to protein misfolding and how to treat those diseases. After reading this unit, you should be able to Describe criteria for measuring protein denaturation Describe the causes for protein denaturation List various diseases occur due to protein folding and misfolding Know the methods to treat protein misfolding
12.1 INTRODUCTION
Protein denaturation is commonly defined as any noncovalent change in the structure of a protein. This change may alter the secondary, tertiary or quaternary structure of the molecules. When using this definition it should be noted that what constitutes denaturation is largely dependent upon the method utilized to observe the protein molecule. Some methods can detect very slight changes in structure while other require rather large alterations in structure before changes are observed.
12.2 MEASURING PROTEIN DENATURATION

12.2.1 Loss of Solubility One of the oldest methods utilized to follow the course of denaturation was to measure changes in solubility. Changes in solubility might be evident in simple buffers or they might exhibit themselves only after exposure to other conditions, eg. 0.25M ammonium sulfate. Proteins vary greatly in their resistance to insolubilization by a variety of procedures and some proteins that are very important in foods are insoluble in their native state. The loss of solubility is only one of the last stages in a series of changes in structure that must have occurred. As such, this is a rather crude measure of protein denaturation. In another sense however, the loss of solubility can be related to the loss of a great number of desirable characteristics of the protein. In many cases in food systems, most structural changes other than loss of solubility are unimportant and the role of many process designs and food additives is to maintain protein solubility. When more sophisticated techniques are utilized many changes in protein structure that eventually result in a loss of solubility can be detected. In these cases the loss of solubility is more properly regarded as an effect of denaturation rather than as a measure of denaturation. To a consumer or a product development scientist who only observes that feathering occurs when some products are utilized to whiten coffee, loss of solubility, however, is the only event that matters. In the rest of this chapter, loss of solubility will be considered as an effect of denaturation.
12.2.2 Increased Proteolysis Most native proteins are quite resistant to the action of proteolytic enzymes. During digestion, proteins are exposed to extremes of pH to alter their structures in such a way as to expose the proper groups to enzyme molecules. For some time, it has been known that a variety of procedures that alter protein's structures make them more susceptible to proteolysis. The rate and extent of proteolysis can be utilized as an indictor of protein denaturation. In many cases, increases in proteolysis, like decreases in solubility, are the result of many changes in protein structure. In a series of experiments on ribonuclease, Burgess and Scheraga exposed this protein to a variety of combinations of pH and temperature. The molecule was then mixed with one of three different proteases. Under conditions of mild denaturation, they were able to observe which portions of the molecule were made susceptible to proteolysis first. Increasingly harsh treatments exposed other portions of the molecule to the action of the proteases. From these observations and knowledge of the tertiary structure of the molecule, they were able to hypothesize a pathway for the thermal denaturation of ribonuclease. This pathway was assumed to be the reverse of the pathway for protein folding, but there was no evidence for this to be the case. 12.2.3 Loss of Biological Activity For those proteins that are enzymes, denaturation can be defined as the loss of enough structure to render the enzyme inactive. Changes in the rate of the reaction, the affinity for substrate, pH optimum, temperature optimum, specificity of reaction, etc., may be affected by denaturation of enzyme molecules. Loss of enzymatic activity can be a very sensitive measure of denaturation as some assay procedures are capable of detecting very low levels of product. In some cases the loss of activity can be shown to occur only after some other changes in structure can be observed by other procedures. There may technically, then be denaturation of the protein before loss of activity occurs. Enzymes are extremely important in the processing and preparing of food products. Processors may variously want to encourage or inhibit the activity of selected enzymes. In these cases, losses of activity may well be the only index of protein denaturation that is of interest. A number of protein molecules may exhibit biological activities that are not enzymatic in nature. Antibodies for instance are capable of interacting with specific antigen molecules. Other proteins, like hemoglobin, may function as carriers while some, eg. Ferritin may function in the storage of specific components. The loss of any of these activities can be measured as protein denaturation. 12.2.4 Tritium-Hydrogen Exchange
When compounds that contain tritium are placed in water they will rapidly exchange the tritium for normal hydrogen if the groups containing the tritium are exposed to the water. Tritium may be incorporated into proteins by a number of procedures. Probably the most common in exchange experiments involves the unfolding of the protein molecule in a medium where all of the water has been replaced by tritium oxide. When the protein is removed to a normal aqueous environment, three classes of tritium are often observed. Any tritium that is on the surface of the molecule along with any other that is not necessarily always on the surface, but that comes into contact with the surface under the conditions of study, will rapidly be lost from the molecule. A second class of tritium molecules will be lost only when conditions that lead to partial protein unfolding occur. These are the class that can be utilized to monitor the rate and extent of denaturation. There may also exist a set of tritium molecules that are located in positions that are accessible to solvent only when the protein molecule is completely unfolded. The second group of tritium atoms do not exchange with the solvent because they are not exposed to the water. Such molecules must be located on the interior of the protein. If denaturation results in unfolding of the molecule and exposure of previously buried tritium groups to the solvent, exchange will occur. This procedure has been utilized quite extensively to study the mechanisms of stabilization of protein structure by small molecules. 12.2.5 Spectroscopic Procedures A variety of procedures have been developed that measure the interaction of electromagnetic radiation with molecules. Some of these procedures have proven to be very useful in the study of protein denaturation. One such procedure is ultraviolet adsorption spectroscopy. This simply measures the wavelength of and the amount of ultraviolet radiation absorbed by a molecule. In proteins, both the wavelength and extent of absorption depend on the amino acids present and on their physical environments. There are a large number of such groups in a protein molecule and thus its U.V. spectrum quite often lacks detail. Under some circumstances however, these groups can absorb at a low wavelength, generally in the U.V., and then emit light at a larger wavelength. This process is known as fluores\cence and is quite sensitive to the environment of the groups involved. Both ultraviolet and fluorescence spectroscopy have been utilized to follow changes in the environments of various groups within protein molecules. Such changes in environment reflect changes in protein structure and thus denaturation. The interaction of polarized light with protein can be measured by the techniques of circular dichroism and optical rotatory dispersion. These methods yield an indication of the extent of repeating structures present in protein and are generally utilized to give estimates of the amount of secondary structure present, eg. Alpha- helix, beat sheet or coil. While these procedures do not yield very precise estimates of the exact secondary structure of proteins, they are very useful for observing changes. These methods are very sensitive and rather small changes in structure can be detected.
12.3 CAUSES OF PROTEIN DENATURATION Changes in the structure of proteins can be caused by a variety of factors. Some of these are encountered frequently while others are more of theoretical interests. Some of the important mechanisms of protein denaturation to food scientists will be discussed. 12.3.1 Thermal Denaturation When proteins are exposed to increasing temperature, losses of solubility or enzymatic activity occurs over a fairly narrow range. Depending upon the protein studied and the severity of the heating, these changes may or may not be reversible. As the temperature is increased, a number of bonds in the protein molecule are weakened. The first affected are the long range interactions that are necessary for the presence of tertiary structure. As these bonds are first weakened and are broken, the protein obtains a more flexible structure and the groups are exposed to solvent. If heating ceases at this stage the protein should be able to readily refold to the native structure. As heating continues, some of the cooperative hydrogen bonds that stabilize helical structure will begin to break. As these bonds are broken, water can interact with and form new hydrogen bonds with the amide nitrogen and carbonyl oxygens of the peptide bonds. The presence of water further weakens nearby hydrogen bonds by causing an increase in the effective dielectric constant near them. As the helical structure is broken, hydrophobic groups are exposed to the solvent. The effect of exposure of new hydrogen bonding groups and of hydrophobic groups is to increase the amount of water bound by the protein molecules. The unfolding that occurs increase the hydrodynamic radius of the molecule causing the viscosity of the solution to increase. The net result will be an attempt by the protein to minimize its free energy by burying as many hydrophobic groups while exposing as many polar groups as possible to the solvent. While this is analogous to what occurred when the protein folded originally, it is happening at a much higher temperature. This greatly weakens the short range interaction that initially direct protein folding and the structures that occur will often be vastly different from the native protein. Upon cooling, the structures obtained by the aggregated proteins may not be those of lowest possible free energy, but kinetic barriers will prevent them from returning to the native format. Any attempt to obtain the native structure would first require that the hydrophobic bonds that caused the aggregation be broken. This would be energetically unfavorable and highly unlikely. Only when all the intermolecular hydrophobic bonds were broken, could the protein begin to refold as directed by the energy of short range interactions. The exposure of this large number of hydrophobic groups to the solvent, however, presents a large energy barrier that make such a refolding kinetically unlikely. Exposure of most proteins to high temperatures results in irreversible denaturation. Some proteins, like caseins, however, contain little if any secondary structure and have managed to remove their hydrophobic groups from contact with the solvent without the need for extensive
structure. This lack of secondary structure causes these proteins to be extremely resistant to thermal denaturation. The increased water binding noted in the early stages of denaturation may be retained following hydrophobic aggregations. The loss of solubility that occurs will greatly reduce the viscosity to a level below that of the native proteins.
2
Fig 51. Protein denaturation
12.3.2 pH Denaturation Most proteins at physiological pH are above their isoelectric points and have a net negative charge. When the pH is adjusted to the isoelectric point of the protein, its net charge will be zero. Charge repulsions of similar molecules will be at minimum and many proteins will precipitate. Even for proteins that remain in solution at their isoelectric points, this is usually the pH of minimum solubility. If the pH is lowered far below the isoelectric point, the protein will lose its negative and contain only positive charges. The like charges will repel each other and prevent the protein from aggregating as readily. In areas of large charge density, the intramolecular repulsion may be great enough to cause unfolding of the protein. This will have an effect similar to that of mild heat treatment on the protein structure. In some cases the unfolding may be extensive enough to expose hydrophobic groups and cause irreversible aggregation. Until this occurs such unfolding will be largely reversible. Some proteins contain acid labile groups and even relatively mild acid treatment may cause irreversible loss of function. This generally results from the breaking of specific covalent
bonds and thus should be considered separately from denaturation. Exposure to strong enough acid at elevated temperatures will first release amide nitrogen from glutamine and asparagine groups and eventually lead to hydrolysis of peptide bonds. The effects of high pH are analogous to those of low pH. The proteins obtain a large negative charge which can cause unfolding and even aggregation. The use of high pH to solubilize and alter protein structure is very important to the formation of fibers from proteins of plant origin. A number of reactions can cause chemical modification of proteins at alkaline pH's that are commonly encountered in protein processing. Many of these involve cysteine residues. Perhaps the most important are the base catalyzed beta eliminations of sulfur to yield dehydroalanine which can react with lysine to form lysinoalanine. This result in a loss of nutritive value of the protein and the products of the reaction may be toxic. Exposure of protein molecules to high pH should be minimized as much as is possible. Exposure to very high pH at elevated temperatures results in alkaline hydrolysis of peptide bonds. 12.3.3 Changes in Dielectric Constant The addition of a solvent that is miscible with water, but that is less polar will lower the dielectric constant of the system. This will tend to increase the strength of all electrostatic interactions between molecules that were in contact with water. Many of the protein hydrogen bonds are effectively removed from the solvent and will not be affected. The presence of the less polar solvent will also have the effect of weakening the hydrophobic bonds of the proteins. These bonds depend upon an increase in the order of water when they are broken for their existence. As there is less water in the system, this becomes less important and at some level of replacement, these groups are at a lower energy level when in contact with the solvent. The structure of the protein will be changed and hence, it will be denatured. The reversibility of the process depends to a large extent on the nature of the non-polar solvent, the extent of unfolding the temperature of the system and the rate of solvent removal. When large amounts of the solvent are present, the protein will be largely unfolded with extensive exposure of the hydrophobic groups. If the protein could be instantaneously transferred to pure water at room temperature, the protein would most likely aggregate and precipitate. The sudden exposure of the hydrophobic groups to water would cause them to try to remove themselves from the aqueous phase as soon as possible. Even before the short range interactions could redirect the folding of the protein aggregation would occur. If the solvent exchange were slow, there would be a better chance that the hydrophobic groups would be able to return to the interior of the molecule and prevent aggregation. If the exchange occurred at low temperatures, the chances of regaining the native structure would be even better. At low temperatures, the hydrophobic groups may in part be stable in the aqueous phase or at least not as unstable. In this case, the removal of the solvent has little affect. When the temperature is subsequently increased, the normal course of protein refolding can occur. Solvent precipitation is often utilized as a means of purifying and concentrating enzymes. It is extremely important that both the solvent and the protein solution be cold when they are mixed
and that the subsequent removal of the solvent is performed at reduced temperature. This helps to insure the recovery of enzyme activity. 12.3.4 Denaturation at Interfaces When proteins are exposed to either liquid-air or liquid- liquid interfaces, denaturation can occur. As a liquid- liquid interface, the protein comes into contact with a hydrophobic environment. If allowed to remain at this interface for a period of time proteins will tend to unfold and place as many of their hydrophobic groups as possible in the non-aqueous layer while maintaining as much charge as possible in the water layer. To understand why proteins unfold at hydrophobic interfaces, it must be realized that the tertiary structure of a protein is not rigid. There are continued fluctuations about an average configuration. Any change in conformation that yields a higher energy state will spontaneously go back to the state of lowest energy. As a part of this process, hydrophobic groups will occasionally be positioned so that they have increased contact with the aqueous phase. When this occurs, these groups will assume the configuration of lowest free energy and will be removed from the water. If a hydrophobic group is exposed while a protein is in contact with a polar solvent, these groups will find a state of lower energy exists if they enter into the solvent phase. This will continue to occur until random fluctuations in protein structure can no longer yield a configuration of lower free energy. The amount of unfolding that occurs at such an interface will depend on how rigid the threedimensional protein structure is an on the number and location of hydrophobic groups in the molecule. A flexible, non-crosslinked protein will be able to unfold easier than will a highly structured and crosslinked one. If energy is applied to cause shear, the process will be accelerated. The shear can cause the protein to unfold, thus exposing its hydrophobic groups to the nonaqueous phase. It can also increase the interfacial area between the two phases and allow more proteins to come into contact with the nonaqueous phase. This unfolding is essentially non-reversible because of the large energy barriers. Even if the phases should separate and the protein is forced into the aqueous phase the protein will not regain its original structure. Rather an association of hydrophobic groups will cause the protein to aggregate. The same forces are in operation when a protein migrates to a liquid-air interface. Hydrophobic groups tend to associate in the air and the protein unfolds. The presence of shear causes to help unfold the protein and to introduce more air into the solution. Both of these effects can be minimized by keeping the temperature low (to weaken hydrophobic bonds) and by minimizing the interfacial area. If the interface is limited, then only a small amount of protein will be able to denature. The presence of this denatured protein will serve as a barrier to further denaturation. Proteins are often utilized in food products to stabilize emulsions or to incorporate air. These cases will be examined in more detail when emulsions and foams are discussed.
12.3.5 Ionic Strength Proteins are usually more soluble in dilute salt solutions than in pure water. The salts are thought to associate with oppositely charge groups in the protein. This combination of charged groups bonds more water than do the charged groups alone and protein hydration is increased. With most proteins there is little change in solubility as more salt is added until some very high salt content is reached. At very high levels of salt there is a competition between the ions and the proteins for water of hydration.
When the salt concentration is high enough, the proteins will be sufficiently dehydrated to lose solubility. Removal of the salt or dilution to a low enough concentration will usually result in the recovery of native structure. 12.3.6 The Effect of Protein Cross linkers The presence of groups that crosslink protein molecules will tend to lower the extent of protein denaturation. There are two main reasons that this is so. First, when proteins are crosslinked it is more difficult for them to unfold. As energy is added to the system and secondary bonds are weakened, the presence of crosslinkers will tend to maintain structure. This is especially true if the crosslinks are covalent as in the case of disulfide bonds. The more compact the molecule is and the greater the number of disulfide linkages present, the greater the stability of the protein. While secondary forces may be weakened and some bonds can be broken, the crosslinkers will tend to keep these groups in fairly close proximity. They also tend to prevent the exposure of large numbers of hydrophobic groups to the solvent. When conditions are returned to the native state, there is now a much greater chance for the proper secondary interaction to occur and for the protein to assume the native configuration. A second effect has to do with the differences in entropy between the native and unfolded states. If a protein can be caused to assume a completely random coil conformation, there will be a large increase in entropy compared to the native structure. This entropy must be overcome if the protein is to refold into a native conformation. When crosslinking groups are present, a completely random coil conformation can not be assumed. These groups introduce order into the structure and there is a considerable loss in the amount of disorder that can be achieved in the most denatured state. Because of this, the entropy change between the native and denatured state is not nearly as great and there will be less of a driving force for denaturation. If the crosslinking groups are broken before denaturation and thus allowed to randomly form after denaturation, no stability will be added to the protein by the pressure of these groups.
Fig 52. Partially folded intermediates at the junction between productive and off-pathway folding. Generalized pathways showing an inclusion body derived from an intermediate on the folding pathway. This illustration shows a speculative intermediate in the formation of a / protein in which a helical domain is docking against a sheet. In the inclusion body pathway, the same interaction proceeds between intermediates, resulting in a polymeric aggregate
12.4 DISEASES RELATED TO PROTEIN FOLDING

12.4.1 Familial Amyloidotic Polyneuropathy Over the past several years, collaborators have conducted similar studies in connection with a human disease. The minor differences between their results and others are very revealing. In the hereditary disease familial amyloidotic polyneuropathy (FAP), peripheral nerves and other organs are damaged by deposits of amyloid-type protein. Although the disorder is quite rare, extensive genetic studies have shown that the disease results from mutations in the protein transthyretin. As with theP22 tail spike protein, transthyretin contains large amounts of -sheet structure and normally consists of several identical amino acid chains (four in this case) associated into a single, three-dimensional structure. FAP results from any of more than 50 distinct mutations within the transthyretin protein, each altering a single amino acid. After studying several of these, scientists found that their four chain structure is less stable under mildly acid conditions than is the wild-type structure. This contrasts with the P22 tail spike mutations, which fold slowly but are stable once folded. It also appears that transthyretin aggregation takes place from a monomeric unfolding intermediate, rather than the folding intermediate involved in P22 tail spike aggregation (the pathway may or may not be the
same in both directions).In both cases, however, the single-chain intermediates have structures that nature has designed for association with other chains of the same type. It apparently takes only a very small change in the shape of these intermediates to alter their normal linkage with two or three other chains into an endless series of linkages that creates insoluble gunk. There is yet another contrast between the P22 tail spike mutations and those in transthyretin: From the P22 viruss view, the problem with the tail spike mutations is that not enough normal protein is made. People with transthyretin mutations, on the other hand, have all the normal transthyretin they need to carryout its usual function (transporting the thyroid hormone). The problem is that, as the protein is being broken down, it forms insoluble gunk, and the insoluble gunk poisons the tissues where it is deposited. 12.4.2 Alzheimers disease FAP is a rare disease; not so Alzheimers, which afflicts 10percent of those over 65 years old and perhaps half of those over 85.Every year Alzheimers not only kills 100,000 Americans, but also costs society $82.7 billion to carefor its victims. In 1991, several different research groups found that individuals with specific mutations in their amyloid precursor protein developed Alzheimers disease asearly as age 40. The body processes amyloid precursor protein into a soluble peptide (small protein) known as A; under certain circumstances, A then aggregates into long filaments that cannot be cleared by the bodys usual scavenger mechanisms. These aggregates then form the -amyloid, which makeup the neuritic plaque in Alzheimer patients. So the consistent association of amyloid precursor protein mutations with early-onset Alzheimers has finally answered a long-debated question: the deposition of neuritic plaque is part of the pathway leading to the disease, not a late consequence of it. To help understand the A aggregation process, researchers chemically synthesized fragments of the 40-amino-acid- long peptide. By using these fragments, they showed that the key step is getting started. Specifically, the precursor fragments have to forma specific nucleus, which then grows into the amyloid process. Possibly the slowness of this first step is why Alzheimers disease is almost entirely limited to older people, and it could be that the mutations in amyloid precursor protein that lead to early-onset Alzheimers are the ones that make it progress more quickly and easily. Even so, A remains soluble in most people. Most individuals who develop Alzheimers disease have the normal form of amyloid precursor protein, indistinguishable from that in people whenever acquire the disorder. Why the same form of A aggregates in some peoples brain but not in others remains a mystery, although a recent discovery has suggested an intriguing possibility. We know that people with different genetic variants of the protein apolipo protein E (apoE) have quite different risks of developing Alzheimers disease. Compared to those with the most common variant, known as apoE3, those with the apoE4 variant are significantly more likely to develop the disease. Some studies suggest that those with the apoE2 variant may be at lower risk, although other studies disagree. These findings are particularly surprising because apoE is best known as part of the complex that transports cholesterol and other fatty materials in the blood stream. What could a fattransporting protein have to do with Alzheimers disease? It may be significant that small amounts of this protein are associated with neuritic plaque and that apoE binds to A in the test tube. The results of this binding are in dispute, however. Researchers report that adding apoE to a test-tube solution of soluble Acauses rapid formation of plaque-type -amyloid fibers and
that apoE4 does so more rapidly than apoE3. Others, however, have obtained opposite results: apoE prevents fibril formation. Thus, whereas some suggest that apoE acts as a pathological chaperone, one that actually promotes misfolding, other researchers believe that it exerts a normal chaperones protective effect. In either case, apoEs influence on the folding of A may play a major role in development of Alzheimers disease. 12.4.3 Mad Cow and Other Species Perhaps the most interesting example of a protein folding disorder is Mad Cow disease and its human equivalent, Creutzfeldt-Jacob disease. These diseases, along with the sheep version known as scrapie, have had the scientific community in an uproar for years. They are infectious diseases transmitted by prions, or protein particles. Prions seem to be pure protein; they contain neither DNA nor RNA. Yet an infectious agent is necessarily self-replicating. How, scientists asked themselves, could a pure protein replicate itself? The answer now starting to emerge may be viewed as a variation on the concept of the pathological chaperone, only in this case the protein serves as its own chaperone.The protein whose aggregation damages nerve cells in Mad Cow disease is constantly being produced by the body. Normally, though, it folds properly, remains soluble, and is disposed of without problem. But suppose that some how a small amount misfolds in a particular way so as to become a scrapie prion. If this scrapie prion bumps into a normal- folding intermediate, it shifts the folding process in the scrapie direction and the protein, despite its perfectly normal amino acid sequence, ends up as more scrapie prion. And the process continues: So long as the body keeps producing the normal protein, a little bit of scrapie prion can keep on creating more and then more. In effect, the prion is replicating itself without needing any nucleic acid of its own. What old-school scientists find even more strange is that the process resembles something akin to genetics. Different strains of these diseases, with somewhat different clinical symptoms breed true as they are transmitted from one animal or human to another. Moreover, these strain differences are associated with slight differences in the protein deposits that apparently cause the disease. (Scientists have recently used these strain differences to show that a few Britons truly have Mad Cow disease, the form seen in cattle, rather than the usual human form of CreutzfeldtJacob disease.)Just as replication can occur without DNA or RNA, other experiments have shown how genetics is possible without nucleic acids. Thus, when researchers mix seed quantities of two different scrapie prion strains in separate test tubes with large amounts of normal protein; each test tube produces more of the specific scrapie prion strain that was added. That is, each strain induces the normal protein to fold in exactly the sameway as the original seed. The strain breeds true in the test tube, just as it does in the body. Odd as it may seem, genetics without nucleic acid is truly possible in the world of protein folding. Despite the examples of FAP, Alzheimers disease, and Mad Cow disease, in which the problem derives from accumulation of toxic, insoluble gunk, many human diseases arise from protein misfolding leaving too little of the normal protein to do its job properly.The most common hereditary disease of this type is cystic fibrosis. Recent research has clearly shown that the many, previously mysterious symptoms of this disorder all derive from lack of a protein that regulates the transport of the chloride ion across the cell membrane. More recently scientists have shown that by far the most common mutation underlying cystic fibrosis hinders the dissociation of the transport regulator protein from one of its chaperones. Thus, the final
steps in normal folding cannot occur, and normal amounts of active protein are not produced .A hereditary form of emphysema shows an even greater analogy to the mutations studies in P22 tail spike protein. Investigators have found that one of the most common mutations producing this disorder greatly slows the normal folding process, just as theP22 temperaturesensitive mutations do. As with the tail spike mutations, the resulting buildup of a crucial folding intermediate leads to aggregation, which deprives affected individuals of enough circulating 1 -antitrypsin to protect their lungs. Emphysema is the result. As intriguing as these examples may be, there is a far more common instance of misfolding, which leaves too little normal protein to do its job. In this case, the proteins job is to block cancer development. Over the past couple of decades, scientists have learned that most cancers result from mutation in the genes that regulate cell growth and cell division. The most common of these genes, involved in roughly 40% of all human cancers, is p53. The sole function of the p53 protein appears to prevent cells with damaged DNA from dividing before the damage is repaired (or to induce them to destroy themselves, if the damage cannot be fixed). In other words, p53 exists to prevent cells from becomingcancerous.p53 mutations associated with cancer fall into two classes. The first keeps the protein from binding to DNA; the other makes the folded form of the protein less stable. In the second group, there is simply never enough properly folded protein around to block the division of DNA-damaged cells. It will be interesting to see how many of the p53 mutants fall into this second class and whether some way can be found to stabilize them.
12.5 TREATING PROTEIN MISFOLDING

The purpose of studying any human disease is to find ways to treat it. The story of protein folding has not yet led to treatments for the diseases involved, but this could happen within the next decade. The key is to find a small molecule, a drug that can either stabilize the normally folded structure or disrupt the pathway that leads to a misfolded protein. Although many molecular biologists and protein chemists believe this will be quite difficult, others are more optimistic.It is difficult to pin point where the search for treatment currently stands, however. One scientist notes that the bulk of that work is tied up in the patent stage: companies are pursuing it but have published little on the subject.Nevertheless, one research group has shown that both thyroidhormone and the related compound TIP (2, 4, 6-triiodophenol) can stabilize transthyretin. SinceTIP neither blocks the action of thyroid hormone nor exerts any hormonelike effects of its own, it appears to be a promising treatment for FAP.Developing smallmolecule therapies is quite straight forward for proteins like transthyretin that naturally bind small molecules,but these therapies are more difficult to apply to proteins that do not have a smallmolecule binding site.One of the few other groups currently publishing their research on smallmolecule structure stabilizers is working to stabilize p53, an acknowledged difficult target. In fact, one laboratory has obtained encouraging results by using two different approaches.Treatments based on our growing knowledge and contined research of protein folding are onthe way. When they arrive, the saga that began with Paulings is fundamental studies of protein structure and Anfinsens investigation of what some call the second genetic code will reach its practical fruition.
12.6 LET US SUM UP Protein denaturation is commonly defined as any noncovalent change in the structure of
a protein. Changes in the structure of proteins can be caused by a variety of factors like thermal denaturation, pH denaturation, changes in dielectric constant, denaturation at interfaces, ionic strength and the effect of protein cross linkers.

Analyze the ways to measure the protein denaturation process.

Review all the ways to measure the protein denaturation process and analyze which one is the best (Refer 2.2).
12.9 REFERENCES
(1) Richard K. Owusu-Apenten (2) Lepock JR. Food Protein Analysis Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry, Methods. 2005 Feb;35(2):117-25. Epub 2004 Dec 19
(3) http://class.fst.ohio-state.edu/FST822/lectures/Denat.htm (4) http://en.wikipedia.org/wiki/Denaturation_(biochemistry)
Lesson 13- MOLECULAR CHAPERONES

Contents: 13.0 Aims and Objectives 13.1 Introduction 13.2 The Role of Molecular Chaperones in Protein Folding 13.2.1 The Link between Translation, Folding, and Membrane Translocation 13.2.2 The Hsp70 Family: Mechanism of a Multipurpose Chaperone 13.2.3 Structure of Hsp70: nucleotide and peptide binding 13.2.4 Function of Hsp70 in protein folding 13.2.5 Function of Hsp70 in membrane translocation of proteins 13.2.6 The Chaperonins: Mediators of Polypeptide Chain Folding 13.2.7 The bacterial chaperonin GroEL: structure and function 13.2.8 Critical features of the chaperonin cycle 13.2.9 Folding in association with GroEL vs. folding in free solution 13.3 Let us sum up 13.4 Check your Progress 13.5 Suggested Readings/References/Sources

In this lesson we have described briefly about the molecular chaperones and the role of molecular chaperons in protein folding. After going through this chapter, you must be able to Understand the roles, structures and mechanisms of molecular chaperones. Describe the link between Translation, Folding, and Membrane Translocation Describe function of Hsp70 in protein folding and in membrane translocation of proteins Describe critical features of the chaperonin cycle.
13.1 INTRODUCTION
In molecular biology, chaperones are proteins that assist the non-covalent folding/unfolding and the assembly/disassembly of other macromolecular structures, but do not occur in these structures when the latter are performing their normal biological functions. The common
perception that chaperones are primarily concerned with protein folding is incorrect. The first protein to be called a chaperone assists the assembly of nucleosomes from folded histones and DNA and such assembly chaperones, especially in the nucleus, are concerned with the assembly of folded subunits into oligomeric structures. Chaperones do not convey steric information required for proteins to fold: thus statements of the form `chaperones fold proteins` are misleading. One major function of chaperones is to prevent both newly synthesised polypeptide chains and assembled subunits from aggregating into nonfunctional structures. It is for this reason that many chaperones, but by no means all, are also heat shock proteins because the tendency to aggregate increases as proteins are denatured by stress. Many chaperones are heat shock proteins, that is, proteins expressed in response to elevated temperatures or other cellular stresses. The reason for this behaviour is that protein folding is severely affected by heat and, therefore, some chaperones act to repair the potential damage caused by misfolding. Other chaperones are involved in folding newly made proteins as they are extruded from the ribosome. Although most newly synthesized proteins can fold in absence of chaperones, a minority strictly requires them. More information on the various types and mechanisms of a subset of chaperones which encapsulate their folding substrates can be found in the article for chaperonins. Chaperonins are characterized by a stacked double-ring structure and are found in prokaryotes, in the cytosol of eukaryotes, and in mitochondria. Other types of chaperones are involved in transport across membranes, for example in the mitochondria and endoplasmic reticulum. New functions for chaperones continue to be discovered, such as assistance in protein degradation and in responding to diseases linked to protein aggregation.
13.2 THE ROLE OF MOLECULAR CHAPERONES IN PROTEIN FOLDING

The Mechanism of Protein folding in the cell remains one of the central problems in biology. The fact that a purified, denatured protein can refold spontaneously into its native 3dimensional conformation in vitro has established that the genetic information required for folding is contained in the linear amino acid sequence of the polypeptide chain. Under cellular conditions, however, the realization of that information requires cellular mechanism for safeguarding partially folded nascent and newly synthesized proteins. These mechanisms are now under intensive investigation, and the class of proteins known as molecular chaperones function as their major players. The term molecular chaperone was coined to describe the function of nucleoplasmin in assembling chromatin. Ellis first applied the term more generally to a range of cellular proteins whose principal role in assembly reactions is the binding of unfolded and partially folded forms of other proteins. The main function of these proteins seems to be in preventing (and possibly reversing) incorrect interactions within and between nonnative polypeptide chains.
According to our current model, protein folding in the cell is ensured by the sequential interaction of nascent and newly synthesized polypeptides with chaperones specific for progressively more ordered intermediates on the folding pathway. As a central feature, these interactions accompany a folding polypeptide from its extended state to the comp act native conformation (in the case of a soluble globular protein).Nascent polypeptides on ribosomes or proteins in transit across intracellular membranes are restricted in folding and interact with chaperones that have the capability to bind extended polypeptides. This results in the prevention of premature (mis) folding until the growing polypeptide is able to fold productively. The major chaperones of this type are the ubiquitous Hsp70 proteins, which act together with members of a family of molecular chaperones homologous to bacterial DnaJ. The interaction of Hsp70 proteins and DnaJ homologues with these polypeptides is governed by a cycle of conformational changes in Hsp70 driven by binding and hydrolysis of ATP. Upon completion of translation or translocation of a folding-competent polypeptide domain, the cycle of substrate protein binding and release by the Hsp70 system may be interrupted when the substrate protein adopts a more ordered conformation that can 1Dgress to the native state. However, for many proteins the completion of folding depends on a molecular chaperone of the chaperonin family, either a chaperonin 60 protein (cpn60) (in bacteria, mitochondria, and chloroplasts) or TriC, a protein complex of the eukaryotic cytosol that accepts partially folded polypeptides. These are large multimeric proteins with the structure of two stacked rings of subunits. Binding of unfolded substrate in the chaperonin cavity prevents loss of folding intermediates due to aggregation. Folding of chaperonin-bound protein is promoted by cycles of polypeptide binding and release driven by coordinated binding and hydrolysis of ATP. These reactions may be enhanced by a specific chaperonin cofactor. Critical elements of this versatile protein- folding system are found in the bacterial cytosol and in all compartments of eukaryotic cells where de novo protein folding is known to occur, including the endoplasmic reticulum (ER). Indeed, the sequential model of chaperone interaction relies on the principle that the ubiquitous presence of these proteins reflects a conserved function in de novo protein folding that is necessary in many different settings. However, protein folding in the ER differs from that in the cytosol in that the requirement for a ring- shaped chaperonin molecule is circumvented. Its function may be taken over by various chaperones such as the membrane-bound calnexin and the soluble Hsp94, which can act downstream of the ER homologue of Hsp70, BiP. We will focus our discussion of chaperone pathways on recent progress toward understanding how folding in the cytosol proceeds in the context with translation. Some major strides have been made during the last year in elucidating the basic structure and function of molecular chaperones, including a crystal structure for the chaperonin protein of Escherichia coil, GroEL, and cryoelectron microscopy of chaperonin complexes with bound substrate protein. These advances have led to various proposals for the mechanism by which GroEL mediates folding. We will discuss the structural and functional studies of GroEL with the intent of clarifying the debate. Even though the structure of the polypeptide binding domain of Hsp70 remains to be fully elucidated, considerable progress has been made in describing the cycle of ATP hydrolysis and peptide binding and release. The demonstration that cytosolic and mitochondrial Hsp70s interact with DnaJ homologues has confirmed the general principle that
Hsp70 action is modulated by partner proteins. The discovery of membrane proteins that bind Hsp70s in the ER lumen and mitochondrial matrix has added the interesting possibility that Hsp70 proteins might even provide motive force during protein translocation. 13.2.1 The Link between Translation, Folding, and Membrane Translocation Chaperone interactions with polypeptides on ribosomes Cytosolic Hsp7Os can be co- isolated with nascent polypeptides extracted from prokaryotic and eukaryotic cells and with ribosome-hound polypeptides from a rabbit reticulocyte cell- free translation. Frydman et al. demonstrated that molecular chaperones can interact sequentially with firefly luciferase chains during translation in such an in vitro system. Nascent polypeptides isolated with ribosomes could be co- immunoprecipitated with antibodies to Hsp70 and Hsp40 (a rabbit cytosolic DnaJ homologue). It is surprising that released nascent chains chromatographed in a large complex containing these two stress proteins along with the TCP ring complex TRiC, the cytosolic chaperonin. Hsp70 binding precedes TRIC binding. These results suggest that the ring complex class of chaperones might promote co-translational folding of protein domains. Hansen et al. have also observed that nascent polypeptides released from reticulocyte ribosomes are found in a high molecular mass complex of >700 KDa that contains numerous proteins, including Hsp70. When these investigators trapped nascent chloramphenicol acetyl transferase (a smaller substrate than the 60 kDa luciferase) on ribosomes, the nascent polypeptide chains became quite resistant to chymotryptic digestion when ATP was removed from the system. Thus, under certain conditions the binding of chaperones might function to inhibit access of protease to the nascent chain. The exact timing of chaperone- mediated protein folding relative to translation is not yet clearly understood. Folding may encompass both co- and posttranslational modes, varying according to the needs of each particular protein. TRIC has been shown to have a more persistent interact ion with newly synthesized actin and tubulin, a reaction distinct from its transient (and perhaps usually co-translational) interaction with firefly luciferase. The bacterial chaperonin GroEL has been shown to interact with newly completed proteins both in vitro and in vivo. Slow-folding proteins have been detected in complexes with GroEL formed in intact cells. In cell- free translations, GroEL has been cross-Linked to newly synthesized pre-beta- lactamase and chioramphenicol acetyl transferase. The important function of heat shock proteins in protein folding can be seen in the prevention of aggregation of newly translated proteins. Such aggregate ion occurs in a strain of E. coli expressing heat shock proteins at severely reduced levels, particularly at high temperature. About 30% of the proteins synthesized in the cytosol are apparently unable to fold in a mutant strain functionally defective in the chaperonin GroEL. However, there is as yet no direct evidence for an interaction between incomplete ribosomebound polypeptide ides and GroEL. This may be an important difference between mammalian and prokaryotic folding systems. Evidence has been provided that there may be an even more intimate link between chaperone action and translation. The ssb-1 and ssb-2 isoforms of Hsp70 i n Sacharomyces cerevisiae coprecipitate with polyribosomes, and mutations in translation elongation factors suppress temperature-sensitive ssb mutants. The direct interaction of chaperones with ribosomal
components may serve to guarantee the availability of these components as soon as a translating chain emerges from the ribosome. Wiedmann et al. have identified a ubiquitous and abundant nascent chain-associated complex (NAC) that interacts with extremely short translating polypeptides before they make contact with other chaperones. NAC is a dimeric protein containing subunits of 33 and 21 kDa that can be crosslinked to nascent chains. It co-purifies with ribosomes in the absence of nascent chains, but can be washed off with high salt. NAC is proposed to form a flexible ribosomal exit site, apparently remaining associated with all newly elongated polypeptide segments until displaced by another binding protein. For example, in the presence of signal recognition particle (SRP), NAC is efficiently displaced from the amino-terminal signal peptides of proteins destined for translocation into the ER. This may provide a generally applicable model: the fate of nascent chains-translation and folding in the cytosol vs. translation arrest and prevention of folding to allow membrane translocation- is in part the result of interactions between nascent polypeptides and an array of chaperone proteins that modulate their folding and targeting within the cell. 13.2.2 The Hsp70 Family: Mechanism of a Multipurpose Chaperone Hsp70s are a family of constitutively expressed us well as stress- inducible proteins of approximately 70 kDa found in almost all organisms. These proteins occur in the cytoplasm, nucleus, endoplasmic reticulum, mitochondria, and chloroplasts of eukaryotic cells. Based on their capability to bind to a wide range of nonnative polypeptide substrates, they participate in a diverse array of functions including protein folding and membrane translocation, the degradation of misfolded proteins, as well as specific regulatory processes. 13.2.3 Structure of Hsp70: nucleotide and peptide binding Hsp70s have a highly conserved amino-terminal ATPase domain of around 45 kDa, followed by an approximately 18 kDa portion that contains the peptide binding site and a more variable region of approximately 10 kDa at the extreme carboxyl terminus. Compartment-specific amino-terminal targeting signals and carboxy-terminal retention signals are appended on this basic structure in Hsp7Os located in cellular compartments other than the cytosol. The crystal structure of the ATPase domain of Hsp70 from bovine brain has been determined to 2.2 ; it reveals an identical fold to two ATPases that are known to undergo conformational changes upon binding ATP, the globular G-actin monomer and hexokinase. ATP binding by Hsp7O is thought to effect a conformational change that is transmitted to the carboxy-terminal domain. It has been proposed that the peptide-binding region of Hsp7O might resemble the peptidebinding domain of the human MHC complex that functions in antigen presentation. Multidimensional NMR results indicate. However, that an 18 kDa fragment of rat cytosolic Hsc7O with peptide-binding activity has a novel secondary structure topology that is not shared by the peptide binding domain of MHC . Several lines of evidence suggest that the binding of polypeptide substrates to Hsp70s occurs via an interact ion between the chaperone and an extended polypeptide segment with a net hydrophobic character. The ER Hsp7O BiP selectively binds heptapeptides enriched in hydrophobic amino acid residues. Using affinity panning of a library of peptides displayed on
bacteriophages to characterize the peptide binding specificity of BiP, Blond-Elguindi et al. found that BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions. They proposed that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. The ability of Hsp70s to bind extended peptide segments has been linked to a function of these chaperones in protecting nascent polypeptides. 13.2.4 Function of Hsp70 in protein folding Binding and release of substrates Hsp70s relies on the modulation of their intrinsic peptide affinity by a cycle o f ATP binding and hydrolysis, which in turn is regulated by interaction with a set of partner proteins. Hsp70 occurs in two affinity states for unfolded polypeptides: the ATP-bound form binds and releases peptide rapidly whereas the ADP-bound form binds and releases slowly. The inter conversion between these two states is catalyzed by peptide binding itself and by auxiliary cofactors, such as DnaJ and GrpE. The basic cycle of Hsp70 action on an unfolded polypeptide can be summarized for the E.coli protein by the following series of steps. DnaJ may bind first to the polypeptide substrate, both in vitro and on the translating ribosome. Through its affinity for DnaK, DnaJ then targets the substrate to DnaK. Polypeptide binds to the ATP-bound form of DnaK, interaction with Dna.J catalyzes the hydrolysis of ATP and stabilizes DnaK in the ADP state. As a result, a ternary complex of DnaJ, DnaK, and substrate polypeptide is formed. The details of the interaction between DnaJ and DnaK in the presence of polypeptide remain to be resolved. In the presence of a third partner, the nucleotide exchange factor GrpE, release of ADP from DnaK occurs and the DnaK/DnaJ complex with substrate is made labile. Substrate is released upon subsequent ATP binding to DnaK. The released polypeptide has the option to either fold, be captured by a downstream chaperone for further folding, or rebind to DnaJ and DnaK. Although mitochondria contain a GrpE homolog that functions similarly to E. coil GrpE, the Hsp70 systems of the eukaryotic cytosol and the ER may be independent of such a factor. This may be due, in part, to differences in the interaction between the eukaryotic DnaJ homologues and Hsp70s. Refolding of purified firefly luciferase by DnaK, Dnu.J, and GrpE has now demonstrated that a protein can progress from the unfolded to the native state during ATP dependent rounds of binding and release by DnaK and DnaJ. Kinetic analysis of the Hsp70 cycle revealed that release of substrate from Hsp70 occurs upon nucleotide binding, preceding the slow hydrolysis step. Monitoring fluorescence changes in a labeled substrate molecule, Schmid et al. demonstrated that both the on and off rates for substrate binding are accelerated when Hsp70 binds ATP. This helps to clarify the role of nucleotide in the reaction cycle; in the presence of ATP, the Hsp70-substrate protein complex is destabilized, but the chance to fIx a new binding site is enhanced as well due to the increased on rate for binding. The previous view that substrate release requires ATP hydrolysis by Hsp70 is explained by the inability of certain ATP analogs to mimic the effect of ATP. However, in the case of E. coil DnaK, addition of the non-hydrolysable analog AMP- PNP to an isolated complex of ADP-DnaK, DnaJ, and substrate protein is sufficient to cause polypeptide release provided that dissociation of ADP is catalyzed by the nucleotide exchange factor GrpE .
Much of the reaction mechanism of lisp70 has been revealed in reactions other than protein folding. The E. coilHsp7O, DnaK, cooperates with DnaJ and GrpE in dissociating certain specific protein-protein interactions. Examples of such activities are the replication of bacteriophage and of plasmids, mini-P1 and mini-F. In contrast to protein folding, in these reactions DnaK and Dna.J act on specific proteins, such as the P protein, that are fully folded but apparently expose chaperone recognition elements similar to those found in unfolded polypeptides. The general principles of the Hsp70 ATPase cycle seem to be preserved in both types of reactions. 13.2.5 Function of Hsp70 in membrane translocation of proteins Some interesting twists on the basic mechanism of Hsp70 action exist in yeast mitochondria and ER. Mitochondrial Hsp70 is required for the efficient postranslational translocation of precursor proteins into the mitochondrial matrix. Binding of mtHsp70 to translocating chains has been detected directly via crosslinking of the chaperone to translocation intermediates spanning both mitochondrial membranes. This interaction is thought to trap the portion of the chain that has reached the matrix, driving translocation by inhibiting its backward diffusion through the translocation channel. MIM44 is a membrane component of the, mitochondrial translocation machinery that specifically binds mtHsp70. The Sec63 translocation protein found in the ER of the budding yeast, S. cerevisiae, also interacts with lumenal Hsp70, BiP. Because both the ER and mitochondrial translocation, synthesis contain membrane-bound components that interact with their respective Hsp70s, it has been suggested that Hsp70 proteins may even act like molecular motors during the translocation process, producing translocating force by binding the polypeptide chain at one site and a membrane-bound component of the translocation machinery at another, then pulling the chain across the membrane via an ATP-dependent conformational change. It may be that for co-translational protein translocation, lumenal Hsp70 has a reduced role. 13.2.6 The Chaperonins: Mediators of Polypeptide Chain Folding The chaperonins are large cylindrical complexes composed of a pair of slacked rings with seven to nine subunits each. Bacterial chaperonin 60 (GroEL in E. coil) and mitochondrial cpn60 are made up of heptameric rings of a conserved abundant 6O kDa subunit whose expression increases upon heat stress. The chloroplast chaperonin rubisco binding protein has the same general architecture but contains two types of homologous subunits. The chaperonin of the eukaryotic cytosol, TRiC, is built of two octameric rings containing a family of 55 kDa proteins with sequence homology to the TCP-1 tail complex protein from mouse. The chaperonin TF55 (for thermophilic factor), a close relative to TRiC, has nine subunits per ring. The cpn6Os in bacteria, mitochondria, and chloroplasts require a partner cpn10 protein, called GroES, in E . c o l i . Eukaryotic chaperonins of the TRiC family are apparently independent of such a factor. Chaperonin protein are important to cellular protein folding and assembly. GroEL was first identified as a host protein required for the assembly of bacteriophage heads and T5 tails. However, the first indication that the chaperonins were directly involved in protein assembly reactions came from studies with the chloroplast enzyme ribulose bisphosphate carboxylaseoxygenase (rubisco). A complex between the newly synthesized large subunit, of rubisco and
the rubisco binding protein was shown to be an intermediate in the assembly of this enzyme from its eight large and eight small subunits. Rubisco subunits in a complex with chaperonin gave rise to the formation of assembled enzyme upon addition of ATP. Isolation of a yeast mutant with a temperature sensitive mitochondrial cpn60 confirmed a general role for the chaperonin in the assembly of organellar proteins. The discovery that chaperonins also mediate the folding of monomeric polypeptide chains such as dihydrofolate reductuse (HDFR) was an important step in the evolution of our current understanding of chaperonin function. Polypeptide chain folding, previously thought to be a spontaneous process, now is known to be the primary function of the chaperonins. The general requirement of GroEL for polypeptide chain folding in the bacterial cytosol was demonstrated in an E. coli strain functionally defective in GroEL. 13.2.7 The bacterial chaperonin GroEL: structure and function The structural analysis of GroEL has provided important insight into the function of the chaperonins and was critic al in shaping a new view of cellular protein folding. GroEL is a tetradecameric complex of identical 57 kDa subunits. Electron microscopic analysis revealed its double-toroidal structure with approximate dimensions of 14 nm in diameter and 16 nm in height. The central cavity of the cylinder, intuitively the most plausible site for polypeptide binding, is 4-6 nm in diameter. The structure of the GroEL tetradecamer has recently been solved by X-ray crystallography. Briefly, each monomeric unit of GroEL is composed of an apical, an intermediate, and an equatorial domain. The equatorial domain is the largest and provides the majority of the subunit-subunit interactions within the heptameric rings and all of the interactions across the equatorial plane of the cylinder. This domain contains the ATP binding site. The intermediate domain is the smallest of the three. It is connected to the other two domains through two short and anti parallel segments, which probably serve as hinges in allosteric conformational changes that occur upon nucleotide binding and hydrolysis. The apical domains form the opening of the central channel. The function of GroEL and its cofactor GroES has been reconstituted in vitro with chemically denatured substrate proteins. 13.2.8 Critical features of the chaperonin cycle Substrate binds within the GroEL cavity as a compact folding intermediate When transferred from denaturant to aqueous solution, extended polypeptides collapse within milliseconds to a compact folding intermediate or molten globule state. GroEL can interact with its polypeptide substrates during or after this stage of collapse, binding them in more or less structured compact conformations. The critical evidence for this came from the finding that GroEL-bound polypeptides have the tryptophan fluorescence and the Property of binding the fluorescent dye ANS typical of molten globule intermediates. Recent findings show that compact - lactalbumin (-LA) intermediates with up to three nonnative disulphide bonds arc bound and that their amide hydrogens show a degree of protection from exchange very similar to that of the free molten globule of -LA. This makes it highly likely that the GroEL-bound protein preserves secondary structure, although these elements are not necessarily more stable than in the free compact intermediate. GroEL-bound polypeptides can cover a range of
conformations, including compact intermediates with considerable tertiary structure. In contrast to Hsp70, the chaperonins recognize the hydrophobic surfaces of folding intermediates rather than specific hydrophobic side chains exposed by an extended p eptide segment. The hydrophobic character of the interaction with chaperonin has been consolidated by the demonstration of salt dependence of binding following the classical Hoffmeister series of salts and by calorimetric measurements. There is general agreement that the substrate-binding face of GroEL is in the central channel within the chaperonin cylinder. Cryoclectron microscopy of an authentic GroEL-substrate complex reveals that the 50 kDa protein malate dehydrogenase is located within the chaperonin channel at the level of the apical GroEL domains. The apical domains of GroEL are poorly defined in the crystal structure, probably the result of a flexible character important to their function. In tile current structural model, the inward- facing surface of these domains is occupied by a number of hydrophobic residues, mutation of which affects substrate binding. This is an elegant solution to the problem of controlled binding and release of a protein folding intermediate. Placement of tile binding surface on the inner face of the ring provides a large interacting surf ace for binding substrate while preventing the formation of chaperone aggregates. Nucleotide-dependent structural changes in the binding face might suffice to drive release and folding of bound substrate. A direct competition of the GroES cofactor for the polypeptide binding sites on GroEL may be significant in mediating substrate release. GroES binds asymmetrically to one end surface of the GroEL cylinder ATP hydrolysis and protein folding by GroEL are coordinated by GroES, a single heptameric ring of 10 kDa subunits with a dome-shaped structure. Binding of GroES to GroEL involves a flexible peptide loop on GroES, the site of several independently derived GroES mutations. Uncoordinated ATP hydrolysis by GroEL leads to the aggregation of certain bound substrate proteins. GroES reduces by 50% the initial ATPase rate of GroEL measured in the absence of unfolded polypeptide while increasing the cooperativity of ATP binding and hydrolysis by GroEL. In the presence of nucleotide, stable complexes form between GroES and GroEL, with a usual 1:1 stoichiometry of GroES to GroEL oligomers. Asymmetrical, bullet-shaped GroEL;GroES particles are seen in negatively stained electron micrographs, with GroES sitting like a cap on one GroEL end surface . Conformational changes in both the GroES-adjacent and the distal ring of GroEL can be observed that are probably of functional importance for the folding reaction. The changes in the GroES-distal ring may reflect a negative cooperative effect of GroES binding that prevents the stable association of a second GroES, although initially both ends of the ligand, free GroEL cylinder are competent in GroES binding. Asymmetry is introduced into the GroEL double-toroid by the preferential binding of ATP to only one ring, which then interacts with GroES. Labeling of GroEL:GroES complexes with [32 P] azido- ATP revealed the preferential binding and hydrolysis of ATP in the seven subunits of GroEL, that arc bound to GroES . After ATP hydrolysis, GroES stabilizes these seven GroEL subunits in a tight ADP state. This explains the half-of- the-sites inhibition of the GroEL ATPase by GroES. The interaction between GroEL and GroES is highly dynamic: the GroEL:ADP7: GroES complex dissociates when seven ATP are hydrolyzed in the GroES-distal toroid coupled to the exchange of the tightly bound ADP in the opposite toroid. The
stabilization of the ADP-bound state of GroEL by GroES and the effect of substrate protein oppose each other. Addition of GroES and ADP may be sufficient to cause protein release from GroEL, at least with substrate proteins such as DHFR. Protein binding in turn destabilizes both ADP binding and the GroEL:GroES complex, thus accelerating the dissociation of GroES from GroEL. (Re) binding of ATP and GroES to the GroEL: polypeptide complex is then thought to coordinate ATP hydrolysis by GroEL for polypeptide release and folding. Cryoelectron microscopy of GroEL:MDH and ternary GroEL:GroES:MDH complexes indeed revealed that substrate protein binds, at least initially, to the GroES distal ring of GroEL . After GroES dissociation and rebinding, substrate protein may then be transiently enclosed within the GroEL cavity by GroES . GroES binding indeed causes a dramatic conformational change in the interacting GroEL subunits, resulting in an outward movement of the apical GroEL domains and an increase in volume of the central ring cavity to a dome-shaped space of 65 x80 . It is conceivable that a bound protein of up to 50 kDa could be displaced into this space for (partial) folding, and there is evidence for the binding of GroES to the substrate-containing toroid of GroEL. Substrate polypeptide would exit the cavity when GroES dissociates upon ATP hydrolysis from in the opposite GroEL toroid. Alternatively, GroES may exert its effect on the folding polypeptide from the opposite GroEL toroid. Whichever mechanism of GroES mediating protein release predominates, in this model protein folding would occur during the ATP hydrolysis-dependent cycling of an asymmetrical GroEL:GroES chaperonin Unit. An opposing view is taken by several groups who stress the role in folding of GroEL:GroES2 particles seen in electron micrographs of chaperonin complexes prepared in slightly alkaline buffer in the presence of high concentrations of magnesium and ATP. It was proposed that 1) such symmetrical, football-shaped complexes represent an obligate intermediate in the chaperonin cycle, 2) folding substrates bind to the outside of the chaperonin ring because the GroEL cavity is inaccessible in these complexes, and 3) the chaperonin cycle of complex formation, and ATP hydrolysis, and complex dissociation is independent of the interaction of substrate protein with GroEL. The functional significance of GroEL:GroES2 complexes spears uncertain, however, in light of recent analyses of the functional stoichiometry of GroEL and GroES . These studies showed that 1) binding of a second GroES occurs only when the normal exclusion of this binding is artificially defeated; 2) the ATP-dependent dissociation of the asymmetrical GroEI.:GroES complex occurs normally under conditions where binding of a second GroES is prevented; 3) association of two GroES molecules precludes the subsequent binding of unfolded polypeptide, strongly supporting the view that substrate has to enter the central cavity of the chaperonin; and 4) substrate protein modulates the interaction between GroEL and GroES, consistent with its reported effect to stimulate the GroEL ATPase. 13.2.9 Folding in association with GroEL vs. folding in free solution There is an ongoing debate as to whether folding mediated by GroEL and GroES occurs in association with the chaperonin or in bulk solution. Martin et al. favor a model in which protein binds to GroEL/GroES in the form of an early compact folding intermediate and then GroES-dependent coordinated hydrolysis of ATP releases substrate in a manner allowing at least partial folding in association with the chaperonin. This may be facilitated by a mechanism in which distinct chaperonin binding elements of the folding polypeptide disassociate at different rates from the internal chaperonin surface and/or by release of bound protein into the
cavity beneath GroES. Some of the released protein molecules progress to the fully native state, whereas some do not complete the burying of hydrophobic binding surfaces in the interior of the molecule and retain the form of GroEL-binding intermediate. Under the conditions of extreme molecular crowding prevailing in vivo, these molecules then rebind to the same GroEL before exiting into the bulk solution, this allows the structural rearrangement of unproductive folding intermediates, followed by another round of release for folding. Horwiela and Lorimer maintain that the single major function of GroEL is the unfolding of kinetically trapped folding intermediates. In their model, unfolded substrate is released from GroEL for spontaneous folding in bulk solution. This population of molecules then undergoes kinetic partitioning; a fraction rapidly adopts a native conformation, whereas the rest fails to progress past kinetically trapped intermediates that are prone to aggregation and retain an affinity for GroEL. Upon rebinding to the chaperonin, these molecules are then unfolded to the initially bound state in preparation for another trial of spontaneous folding. This proposal is based on the observation that substrate protein can be released from GroEL, in the presence of ATP and GroES, in a nonnative conformation that can be captured by a noncycling trap of mutant GroEL. In both of these models multiple rounds of ATP-dependent release and rebinding are necessary for complete folding. The crucial mechanistic distinction lies in the question of whether the polypeptide must undergo a period of release into bulk solution before rebinding to another GroEL for successful folding to occur. The observation of partitioning of nonnative folding intermediates between different chaperonin molecules does not address to what extent folding might occur in association with the chaperonin. The mutant GroEL trap persists in a highaffinity binding state for folding intermediates. This might drive the accumulation of highly unfolded molecules, underestimating the degree to which bound molecules have progressed toward the native state upon release from wild-type chaperonin.
The view that the interaction of a polypeptide with GroEL only mediates unfolding of kinetically trapped intermediates would not explain the apparent progression of GroELassociated rhodanase toward a more compact state during a short pulse of ATP hydrolysis in the presence of GroES. It also does not provide an explanation for the findings that rhodanese aggregates upon ATP-dependent release from GroEL, in the absence of GroES or that rhodanese must interact with GroEL throughout most of its GroES-dependent folding reaction. Finally, addition of GroEL to refolding barnase slows refolding; indicating that barnase interacts with GroEL, but even at a high molar excess GroEL cannot abolish barnase folding. It was concluded that for weakly interacting substrates, folding occurs in contact with GroEL. A similar reaction could certainly occur for the parts of a strongly interacting substrate that are not directly involved in tight binding or when ATP binding to GroEL loosens the interaction with the associated polypeptide. A function in unfolding kinetically trapped folding intermediates and a function in mediating productive folding are not mutually exclusive. In fact, the possible reversal of unproductive interactions within folding intermediates upon rebinding to GroEL was envisioned in early models for GroEL mechanism, based on the fact that multiple rounds of release and rebinding are often necessary for successful folding and on the observation of partial unfolding of a GroEL-bound folding intermediate upon inhibition of ATP hydrolysis.
13.3 LET US SUM UP

Many chaperones are heat shock proteins, that is, proteins expressed in response to elevated temperatures or other cellular stresses. Chaperonin protein are important to cellular protein folding and assembly. Uncoordinated ATP hydrolysis by GroEL leads to the aggregation of certain bound substrate proteins.

Most of the chaperons are beat stock proteins Discuss.

Study the role of molecular chaperones in protein folding (Refer 3.2)

(1) Mayer, MP and Bukau, B Hsp70 chaperones: cellular functions and molecular mechanism, Mol Life Sci 62: 670-684, 2005 Structure of an Hsp90-Cdc37-Cdk4 complex, "Mol Cell" 23(5): 697-707, 2006
(2) Vaughan CK, Gohlke U, Sobott F, Good VM, Ali MM, Prodromou C, Robinson CV, Saibil HR, Pearl LH (3) Ellis J
Proteins as molecular chaperones". Nature 328 (6129): 378-9 Protein folding in the cell. Nature 355, 33-45.
(4) Gething, M.J. & Sambrook, J.
(5) http://en.wikipedia.org/wiki/Chaperone (6) http://people.cryst.bbk.ac.uk/~ubcg16z/hsplec.html
Lesson 14 - MOLECULAR FORCES IN RELATION TO PROTEIN STRUCTURE Contents: 14.0 Aims and Objectives 14.1 Introduction 14.2 Non-Bonded Interactions 14.3 Electrostatic Interactions 14.3.1 Salt Bridges 14.3.2 Hydrogen Bonds 14.3.3 Partial Charges 14.3.4 Induction 14.4 Dispersion 14.5 Repulsion Term 14.6 The Lennard-Jones Potential and Van Der Waals Radii 14.7 Hydrophobic Interactions 14.8 Let us sum up 14.9 Lesson End Activities 14.10 Check your Progress 14.11 References

This section of the unit concentrates on the nature of the interactions which underlie the structure of proteins. An attempt will be made to explain the fundamental principles for each effect and the mathematical forms which are conventionally used in computational representations of biomolecules. At each stage implications for protein structure are pointed out.
14.1 INTRODUCTION
It is conventional to talk of the different classes of interactions which act between atoms as "forces" (e.g., the van der Waals force). This can be somewhat confusing as it is normal to go on and give an equation for the potential energy of the particular interaction. In this section an attempt is made to explain the various interactions occur between atoms in a molecule.
14.2 NON-BONDED INTERACTIONS

As the name implies non-bonded interactions act between atoms which are not linked by covalent bonds. Like most things this is simple to state but can be confusing to apply in practice! In most approaches then atoms which are involved in a bond angle are also not regarded as having a non-bonded interaction. 1-4 interactions (those between the end atoms involved in a dihedral angle) are sometimes given an additional scaled down nonbonded interaction. Similarly the interaction between a metal ion and its liganding atoms is usually regarded as non-bonded and treated by the kind of approach set out here but are sometimes represented by the bond and bond angle terms of the potential energy function (e.g., in representing the iron atom in the haem group found in globins).
14.3 ELECTROSTATIC INTERACTIONS

Electromagnetic interactions dominate on the molecular scale and provide the fundamental basis for all the different bonded and non-bonded interactions discussed here. This is clearest in the case of electrostatic interactions where charges on nuclei and electrons interact according to Coulomb's law:
where and are the magnitude of the charges, is their separation, the permittivity of free space and the relative dielectric constant of the medium in which the charges are placed .The strictly correct way to use the law would be to consider every nucleus and electron separately, plug it into the Schrdinger equation and apply quantum chemical methods to solve the
equation for the spatial configuration of nuclei we are interested. As already mentioned this is completely impractical for biomolecular systems. So instead we wish to develop a useful model for the interactions between nuclear centres without having to explicitly deal with the electrons in a system. The simplest approach is to just to consider the formal charges of the protein. Formal charges show whether chemical groups are ionized i.e., whether an atom or set of atoms has lost or gained an electron. Isolated amino acids (in neutral solution) are zwitter ionic - this means that although the molecule has no overall charge it carries both a negatively charged group and a positively charged group:
Fig 53.An isolated alanine residue in solution
Note that the atoms in a charged carboxylic acid are equivalent - the double bond is delocalized across the group. In proteins the individual amino acids are polymerized (in a condensation reaction - releasing water). This results in a peptide backbone which is electrically neutral with the exceptions of the ends of the chain. In a normal protein the amino end carries a positive charge (-NH2+) and the carboxyl end carries a positive charge (-CO2-). In some cases (e.g., a number membrane polypeptides) the ends are chemically modified to avoid these charges (for instance by acetylation of the amino end group). Most of the standard amino acids found in proteins have uncharged side chain groups. However, there are a number of basic residues which are positively charged at normal pH.
Fig 54.
In addition histidine is normally charged at neutral pH (the charge normally residing on the delta carbon but sometimes on the epsilon). When the residue is placed in a basic environment it loses a proton and becomes uncharged:
Fig 55.
There are two standard residues which normally carry a negative charge: glutamic and aspartic acids:
Fig 56.
Very many proteins bind inorganic ionic species such as metal ions - indeed such ions can play a crucial role in the mechanism of a protein. An example of this is the enzyme xylose isomerase which has two Mg2+ ions at the active site which coordinate to the substrate, polarizing it and stabilizing the transition state in the reaction. 14.3.1 Salt bridges As might be expected a positively charged lysine or arginine residue can form a strong interaction with a negatively charged asp or glu group. In proteins this interaction is referred to as a salt bridge. In practice salt bridges are relatively rare in proteins and in practice they normally occur on the surface as opposed to internally. An exception is when an internal salt bridge is involved in the catalytic mechanism of an enzyme such as in the asp-his-ser triad of serine proteases (a classic example of the structural basis of enzyme activity). The reason for this is that although an internal salt bridge is a strong interaction in comparison to having the isolated residues widely separated in a vacuum it is normally destabilizing for a protein. This apparent paradox is due to that fact that when considering the effect of an interaction one must consider the difference in the (free) energy between the folded and unfolded but solvated states. In the unfolded state the residues involved in a salt bridge would be widely separated but each making very favorable interactions with water molecules (for advanced students - there is an entropic contribution to this). These interactions are lost when the same residues are buried in the largely hydrophobic core of the protein. Similar arguments apply to practically all considerations of elucidation of the energetic contributions to protein folding or ligand binding - normally a small overall free energy advantage arises from the balance between large but canceling contributions. This is one of the problems which make the computation prediction and analysis of protein behaviour so difficult. Disulphide Bridges
Fig 57. Disulphide bond
If two cysteine sidechains are close to one another and the local environment is conducive to oxidation, then they can form a disulphide bond/bridge as shown. The residues can either be in the same peptide chain (forming a loop) or in different chains. As Disulphide bonds are covalent linkages they add considerably to the stability of proteins in which they occur. However they mainly occur in secreted proteins and in the parts of membrane proteins which face the outside. Note that disuphide bridges only form after the protein has folded into its tertiary structure. This folding is driven by the combined energy of all of the weak interactions described in the previous section above. Cysteine side chains in cytosolic proteins are usually found as -SH groups as the cytosolic environmemt are sufficiently reducing to prevent disulphides from forming. 14.3.2 Hydrogen Bonds The electrostatic interactions between groups which carry no formal overall electrical charge are of fundamental importance to bimolecular structure. The source of these is that uncharged species can still have a large inherent polarization - the orbital around the molecule are distributed in such a way that parts of the molecule have less electrons and thus carry a positive charge and other parts have an excess and are therefore negatively charged. Some atoms (O, N and to a lesser extent S) have a tendency to attract electrons (filling the valence shells) and are termed electronegative. Others (notably metallic atoms) have a tendency to lose electrons. In extreme this tendency causes one atom completely to lose an electron to another - leading to the formation of formally charged species or ions (see previous section). In less extreme conditions electrons are shared between two atoms in a covalent bond but are pulled towards one partner. The classical example of this is the water molecule: As oxygen is electronegative it draws the electrons in the bonds it shares with the hydrogen atoms towards it. The hydrogen atoms are left with a net positive charge and the oxygen is negative. In the case of a water molecule the value for the effective charge on each hydrogen atom is quite large - around one third of an electron. Together with the short distance between the oxygen and hydrogen atoms this results in the water molecule having a large dipole moment. Two water molecules can therefore form a strong electrostatic interaction:
Fig 58. Hydrogen Bond
This interaction is known as a hydrogen bond (incidental note the lowest energy water dimer is not flat there is an angle of around 23 degrees between the planes of the molecule). The bond is normally around 2.8 long (measured from oxygen to oxygen). This length results from the interplay between the electrostatic stabilizing factor and the repulsion between the oxygen atoms as they come closer. Water molecules can form a network of hydrogen bonds. The fact that it is strong has a number of important consequences: You may remember from high school chemistry this is the reason why water is a liquid at room temperatures whereas hydrogen sulphide (H2 S) is gaseous - despite having a higher molecular weight. Sulphur atoms are less electronegative and sulphur hydrogen bonds are longer so that the dipole moment (degree of inherent polarization) is lower. This has the consequence that although H2 S can make favourable interactions in a manner similar to that above they are much less strong - resulting in less cohesion and a lower boiling point. The anomalous freezing property of water - that ice is less dense than liquid water also results from hydrogen bonding in that ice (where each molecule has to be involved in 4 hydrogen bonds with its neighbours) has a more open structure than water. Despite being called a "bond" a hydrogen bond is very much weaker than the covalent bonds which hold together organic molecules (such as proteins). A typical value for the stabilization of a hydrogen bond is around 6 kcal/mol compared to hundreds for covalent bonds. This results in it being relatively easy to make and reform hydrogen bonds without any need for energy inputs or catalysis. For instance in liquid water molecules are constantly swapping hydrogen bond partners. Furthermore in proteins hydrogen bond patterns can change - for instance on binding. However, in the laboratory it is important to remember that the hydrogen bond can be a strong interaction. You may notice organic solvents containing chlorine atoms are usually stored separately from others awaiting disposal. The reason can be appreciated by considering the interaction between chloroform and acetone:
Fig 59. Interaction between chloroform and acetone
Both chlorine and oxygen are very electronegative so the effective charges on the hydrogen (caused by the chlorines drawing electrons from the carbon) and oxygen are high. Note there is essentially one centre for positive charge on chloroform but that the negative charge is distributed between a number of atoms. The opposite is true for acetone. When the two are mixed they can form a stronger mutual interaction than separately. This results in a fairly exothermic reaction which can heat the system to such an extent that it spontaneously combusts! Hydrogen bonds are of crucial importance to protein structure. The ability of main chain carbonyl oxygen bonds to form hydrogen bonds with the main chain amino groups leads to the possibility of forming different secondary structures, such as alpha helices and beta sheets:
Fig 60. Hydrogen bond in alpha helix
Note that under aqueous conditions the main chain carbonyl and amino groups are stabilized by hydrogen bonds to water even in the unfolded (random coil) state. In the folded state these groups are almost invariably involved in hydrogen bonds but overall there is normally a slight
destabilization in comparison to the random coil (proteins have marginal stability due to effects such as the hydrophobic effect). Many side chain groups in proteins can form hydrogen bonds.In addition interactions can be formed between groups carrying a formal charge and hydrogen bonding .This is normally regarded as an especially strong variant of the hydrogen bond. 14.3.3 Partial Charges We have seen that electrostatic interactions are of fundamental importance to proteins. We shall now briefly examine the manner in which they are normally treated in computational studies. The most common approach is to place a partial charge at each atomic centre (nucleus). These charges then interact by Coulomb's Law. The charge can take fractions of an electron and can be positive or negative. Charges on adjacent atoms (joined by one or two covalent) bonds are normally made invisible to one another - the interactions between these atoms being dealt with by covalent interactions. Note that the concept of a partial charge is only a convenient abstraction of reality. In practice many electrons and nuclei come together to form a molecule partial charges give a crude representation of what a neighbouring atom will on average "see" due to this collection. The standard modern way to calculate partial charges is to perform a (reasonably high level) quantum chemical calculation for a small molecule which is representative of the group of interest (e.g., phenol is considered for tyrosine). The electrostatic potential is then calculated from the orbitals obtained for many points on the molecular surface. A least squares fitting procedure is then used to produce a set of partial charges which produce potential values most consistent with the quantum calculations. Older procedures used methods in which orbital populations are simply split between atoms (Mulliken Population Analysis). All though much simpler these charges do not produce a reasonable representation of the electrostatic potential around a molecule - which is usually what is of interest in a simulation.To see the kind of values typical for partial charges look at picture of a salt bridge . As mentioned previously using partial charges at nuclear centres is the crudest effective abstraction. To obtain a more accurate representation two approaches are common. The first is to add dipole, quadrapole and higher moments to the nuclear centres (do not worry if these terms are unfamiliar.The second is to introduce further non-nuclear centres - this is commonly done to represent the anisotropy in potential cause by lone pairs on oxygen atoms.In many respects electrostatic interactions provided the biggest problems to computational studies of protein behaviour. By their nature they are long range and dependent on the properties of the surrounding medium. A simple rule of thumb is that the more highly charged a system the harder it is to simulate - thus simulations of liquid argon can do a wonderful job, hydrocarbons are fairly easy, water becomes difficult and proteins more so. The limit is reached with nucleic acids like DNA which are aqueous complex salts (each base having a charge of minus two)
with counter ions and solvent having important effects on structures. Usually some sort of "fudge" has to be made in simulations to keep DNA stable at all! 14.3.4 Induction The normal treatment for partial charges is to assume they are fixed. In practice the electric field caused by other atoms and molecules will polarize an atom effecting its electron distribution and thus its partial charge. In turn the partial charge produces an electric field which affects neighbouring charges and thus fields. To be able to work out the partial charges a self consistency cycle is normally used. The process of polarization has an energetic effect. In practice it is difficult to find adequate parameters to treat systems as complex as proteins (work has recently been concentrating on systems such as sodium ions in water). Induction effects can be shown to decay by an r -6 relation so they can normally be regarded as implicitly corrected for when the dispersion term is fitted as discussed in the next section.
14.4 DISPERSION
You will probably be familiar that at low temperatures gases such as argon liquefy. The attractive interactions which cause this are called dispersion. Although they also occur between charged atoms they are usually overwhelmed by the stronger electrostatic terms and so are normally only of importance for uncharged groups. To really understand dispersion effects one must turn to 2nd order perturbation theory in quantum mechanics. You will probably be happy to know that we shall not be doing this! Instead a simple physical picture will be given. Imagine that we have an atom of argon. It can be considered to be like a large spherical jelly with a golf ball embedded at the centre. The golf ball is the nucleus carrying a large positive charge and the jelly represents the clouds of electrons whizzing about this. At a point external to the atom the net average field will be zero because the positively-charged nucleus' field will be exactly balanced by the electron clouds:
Fig 61.
However, atoms vibrate (even at 0K) and so that at any instant the cloud is likely to be slightly off centre. This disparity creates an "instantaneous dipole":
Fig 62.
Suppose that we have another argon atom close to the first. This atom will see the electric field resulting from the instantaneous dipole. This field will effect the jelly inducing a dipole:
Fig 63.
The two dipoles attract one another - producing an attractive interaction.The Dispersion interaction can be shown to vary according to the inverse sixth power of the distance between the two atoms:
The factor Bij depends on the nature of the pair of atoms interacting (in particular their polarizability). It is normal to parameterize the dispersion empirically using structural and energetic data from crystals of small molecules. It is not possible to use simple quantum chemical calculations to find parameters. This is because most quantum chemical calculations use the self consistent field approximation (SCF). In this each electron is solved independently keeping the other orbitals frozen (in a self consistency). This effectively means that electrons only experience a time averaged picture of other electrons - so that dispersion cannot come into effect. More advanced methods in quantum chemistry introduce methods to tackle "electron correlation" to avoid this problem.
14.5 REPULSION TERMS

When two atoms are brought increasing close together there is a large energetic cost as the orbitals start to overlap. In the limit that the atomic nuclei where coincident the electrons of the two atoms would have to share the same orbital system. The Pauli exclusion principle states that no two electrons can share the same state so that in effect half the electrons of the system would have to go into orbitals with energy higher than the valence state. For this reason the repulsive core is sometimes termed a "Pauli exclusion interaction". The simplest and oldest way to represent the repulsive core for atoms is by using a "hard sphere" model. In this atoms have a characteristic radius (below the van der Waals radius) and cannot overlap. This approach can be adopted computationally but is more commonly seen in physical models using plastic spheres such as CPK: This "all or nothing" approach is useful but rather crude - both solids and liquids are compressible. It also leads to the problem that it has a discontinuous first derivative: When two atoms come slightly too close together they experience an infinite force. More realistic is to represent the energy costs of close approach using a term which varies as R-12:
The repulsive term (it is always positive) drops away dramatically as the distance between the two atoms increases but conversely becomes very large at short distances - providing a "fuzzy" core. This is the approach normally adopted for protein potential energy functions. When more accuracy is required a two parameter model is normally adopted:
This term together with a representation for dispersion by a term in Rij-6 is commonly known as the "Buckingham potential". It provides a more realistic representation particularly at short distances where a term in R-12 is too stepped. It is not normally used for macromolecular applications as the increase in complexity (introduction of an additional parameter) and in computational cost (it takes a long time to calculate exponentials) is undesirable.
14.6 THE LENNARD-JONES POTENTIAL AND VAN DER WAALS RADII

The dispersion and repulsion terms discussed above are commonly grouped together into the Lennard-Jones or 6-12 potential:
The equation can be rewritten in an equivalent more instructive form (choosing the case for an interaction between two atoms of the same type):
The minimum of the function is at r = 2R* and has an energy of minus E* . The distance R* is known as the van der Waals radius for an atom and E* is its van der Waals well depth. Typical values for these parameters (from the AMBER force field) are shown below.
Table 4:
It is important to note that the Lennard-Jones interaction between uncharged atoms (such as CH3 groups) is less attractive than that between charged groups such as oxygens. The difference is that the contribution from electrostatics will dominate the L-J interactions. In cases where uncharged groups form compact structures van der Waals energies are often cited as stabilizing the conformation. Although partly true very often the major contribution comes rather from hydrophobic exclusion.
14.7 HYDROPHOBIC INTERACTIONS

The association of relatively non- polar molecule or group in aqueous solution with other nonpolar molecule rather than with water is termed as hydrophobic interaction. Although hydrophobic interactions are sometimes called hydrophobic bonds, this description is incorrect. Non-polar molecules are groups tend to aggregate not because of mutual attraction
but because the polar water molecules trapped around the non-polar compounds tend to associate with each other. For example micelles are stabilized by hydrophobic interactions. The hydrogen- bonding pattern of water is disrupted by the presence of non-polar molecule. Water molecules that surround a less polar molecule in solution are more restricted in their interactions with other water molecules. These restricted water molecules are relatively immobile, or ordered, in the same way that molecules at the surface of water are ordered in the familiar phenomenon of surface tensions. However, water molecules in the bulk solvent phase are much more mobile, or disordered. In thermodynamic terms, there is a net gain in the combine entropy of the solvent and the non-polar solute when the nonpolar groups aggregate and water surrounding the nonpolar groups are freed from its ordered state. Hydrophobic interactions, like hydrogen bonds, are much weaker than the covalent bonds. For example, the energy required to transfer a -CH2- group from a hydrophobic to an aqueous environment is about 3kj mol-1.
Fig 64.
The cumulative effect of many hydrophobic interactions can have a significant effect on the stability of a macromolecule. The three dimensional structure of most proteins, for example, is largely determined by hydrophobic interactions formed during spontaneous folding of the polypeptide chain. Water molecules are bound to the outside surface of the protein but cant penetrate the interior, where most of the non-polar groups are located. All of the interactions covered here are individually weak compared to covalent bonds, but the combined effect of many such weak interactions can be significant.
14.8 LET US SUM UP

In electrostatic interaction charges on nuclei and electrons interact according to Coulomb's law
Salt bridges are relatively rare in proteins and in practice they normally occur on the surface as opposed to internally. As Disulphide bonds are covalent linkages they add considerably to the stability of proteins in which they occur The ability of main chain carbonyl oxygen bonds to form hydrogen bonds with the main chain amino groups leads to the possibility of forming different secondary structures, such as alpha helices and beta sheets The Dispersion interaction can be shown to vary according to the inverse sixth power of the distance between the two atoms The repulsive term (it is always positive) drops away dramatically as the distance between the two atoms increases but conversely becomes very large at short distances providing a "fuzzy" core Non-polar molecules are groups tend to aggregate not because of mutual attraction but because the polar water molecules trapped around the non-polar compounds tend to associate with each other. For example micelles are stabilized by hydrophobic interactions. The hydrogen- bonding pattern

Compare and Contrast Electrostatic and Hydropholic interaction.

Discuss the effect of hydropholic interaction. (Refer 4.7).
14.11 REFERENCES
(1) A. J. Stone (2) Jacob N. Israelachvili The Theory of Intermolecular Forces Intermolecular and Surface Forces: With Applications to Colloidal and Biological Systems
(3) www.biochemistry.bham.ac.uk/osmart/course/os_molf.html (4) http://www.wwnorton.com/college/chemistry/gilbert/tutorials/ch9.htm
Lesson 15 HELICES AND TURNS Contents: 15.0 Aims and Objectives 15.1 Introduction 15.2 Types of Helices 15.2.1 Alpha Helix 15.2.2.3.10 Helix 15.2.3 Pi-Helix 15.3 Tight Turns 15.3.1 Gamma Turns 15.3.2 -Turn 15.4 Let us sum up 15.5 Lesson end Activities 15.6 Check your Progress 15.7 References

In this lesson we have described briefly about the helices, turns and their role in protein folding and stability After going through this chapter, you must be able to: Explain geometry and hydrogen bonding in alpha helix Explain helical axis and stability of alpha helix Role of turns in protein folding
15.1 INTRODUCTION
An alpha helix is a common structure of proteins, characterized by a single, spiral chain of amino acids stabilized by hydrogen bonds. A turn is an element of secondary structure in proteins.According to the most common definition, a turn is defined by the close approach of two C atoms (< 7 ), when the
corresponding residues are not involved in a regular secondary structure element such as an alpha helix or beta sheet.
15.2 TYPES OF HELICES

Helices were the second structural element of proteins to be identified. In a helical conformation, the relationship of one peptide unit to the next is the same for all alpha-carbons. This means that the dihedral angle pairs phi and psi (phii, psii) are the same for each residue in the helical conformation. Helices are classified as repetitive secondary structure since their backbone phi and psi angles repeat (for the geometrically ideal, right-handed, alpha helix, these values are phi = -57.8 and psi = -47.0). The converse is also true i.e. if the backbone dihedral angle pairs are the same for each residue, the resulting conformation will assume a helical conformation about some axis in space. Two parameters describe the helix about this axis: n - the number of residues per helical turn r - the rise per helical residue By convention, a positive value of n denotes a right-handed helix. (Curling the fingers of your right hand along the helical path, your thumb will point in the direction of your fingertips if the helix is right-handed.)
Table 5: Parameters for common proteins helices. Values are given for pure geometrical forms of these conformations. Here n is the number of residues per helical turn, r is the helical rise per residue, and p is the helical pitch (/turn).
Historically (Brag, Kendrew & Perutz, 1950), helices were often designated by the number of residues per helical turn and the number of atoms in one hydrogen-bonded ring This may be given down the page aas a footnote. Thus the alpha, 3.10, and pi helices were designated 3.6(13), 3.0(10), and 4.4(16), respectively. This nomenclature persists only for the 3.10 helix since it can lead to ambiguities (e.g. 3.6(13) and 3.7(13) are both alpha helices). A word of caution in viewing 2-dimensional stereo pictures: There exist two ways of viewing stereo pictures cross eye and wall eye. To be stereochemically accurate, stereo pictures need to be produced and viewed with the same convention. If not, the resulting picture still appears in stereo, but the stereochemical designations are reversed.
Helices are the most abundant form of secondary structure containing approximately 32-38% of the residues in globular proteins (Kabsch and Sander, 1983). Of all possible geometric forms, the alpha helix is by far the most abundant. Short segments of 3.10 helix are found. As we shall see, ends of alpha helices can contain a single 3.10- or pi- like hydrogen bond. The gamma helix was also predicted to be a possible structural element in proteins by Pauling and Corey. This helix has the designation 3.6(14) and was never observed. It may be an interesting exercise to create this very different helical structure (pencil and paper are sufficient if models are not at hand), once you have had a chance to look over this section. You will discover (if you had not already) two very different families of possible helices from polyamino acids which differ in the number of residues in the hydrogen-bonded loop: (3n + 4) and (3n + 5).
Fig 6 5 : Three regular polypeptide helices. A cartoon of a icosapeptide in a right-handed alpha-helical conformation is shown on the far left. Idealized model alpha-helical (phi = -57.8, psi = -47.0), 3.10-helical (74.0, -4.0), and pi-helical (-57.1, -69.7) coformations of a polyalanine icosapeptide are displayed (without hydrogens) using CPK representations. Views are perpendicular to the helical axis with the N-terminal at the bottom (lower) and following a 90 rotation (top). Standard CPK color is used with the exception that the alanine side chain (CB) carbon is a lighter shade of grey for distinction from backbone carbons. All structures are reproduced at the same scale using the program RasMol.
15.2.1 alpha helix The alpha helix is the most abundant helical conformation found in globular proteins accounting for 32-38% of all residues (Kabsch & Sander, 1983; Creighton, 1993). The average length of an alpha helix is 10 residues as taken from surveys of this structural database. The
average dihedral angles phi and psi (-64 +/- 7, -41 +/- 7) also obtained from these surveys are found to differ slightly from the geometrically pure alpha helix (-57.8, -47.0). The abundance of this particular form of secondary structure stems from the following properties: the phi and psi angles of the alpha helix (lie in the center of an allowed, minimum energy region of the Ramachandran (phi, psi) map. the dipoles of hydrogen bonding backbone atoms are in near perfect alignment. the radius of the helix allows for favorable van der Waals interactions across the helical axis. side chains are well staggered minimizing steric interference. A good look at a geometrically pure alpha helix is afforded by the CPK representation shown in Figure 76. Notice how all amide protons point toward the N-terminus (down) and all carbonyl oxygens point toward the C-terminus (up). The repeating nature of the phi, psi pairs ensure this orientation. Hydrogen bonds within an alpha- helix also display repeating pattern in which the backbone C=O of residue i hydrogen bonds to the backbone HN of residue i+4. Looking at the helix along the helical axis from the C-terminus (top), you can see the four carbonyl oxygens of the last turn of the helix and the dispersion of sidechains. Residues in positions (i, i+3) and (i, i+4) are positioned in such a way as to force interaction of their sidechains. This can have a stabilizing effect if the residues are of opposite charge or are both hydrophobic. Interaction between aromatic rings (Phe) at position (i) and His at position (i+4) appears to have a stabilizing effect on the helical conformation of the C-peptide of ribonuclease in solution (Armstrong et al., 1993). 15.2.2 3.10 helix The 3.10 helix is not a common secondary structural element in proteins. Only 3.4% of the residues are involved in 3.10 helices in the Kabsch and Sander database (1983), and nearly all those in helical segments containing 1-3 hydrogen bonds (96% less than or equal to 4 residues). Alpha helices sometimes begin or end with a single turn of a 3.10 helix (one hydrogen bond) although this accounts for <25% of residues with this classification. The average of the backbone dihedral angles were found to differ slightly from the ideal 3.10 helix (-74.0, -4.0) with values of -71.0 and -18.0 degrees, for phi and for psi, respectively. This seemingly small difference results in a larger radius (2.0 versus 1.9 ) and a larger number of residues per helical turn (3.2 versus 3.0). The end result being a slightly better staggering of sidechains along the helical axis. Hydrogen bonds within an 3.10-helix also display a repeating pattern in which the backbone C=O of residue i hydrogen bonds to the backbone HN of residue i+3. The infrequency of this particular form of secondary structure stems from the following properties: the phi and psi angles of the pure 3.10 helix (-74.0, -4.0) lie at the edge of an allowed, minimum energy region of the Ramachandran (phi, psi) map. the dipoles of hydrogen bonding backbone atoms are not well aligned with the carbonyl oxygens splayed out away from the helical axis (~30 degrees). van der Waals contact across the helical axis may cause repulsion. side chains are in identical azimuthal positions leading to potential steric
interference. A good look at a geometrically pure 3.10 helix is afforded by the CPK representation shown in Figure 76. Again notice how, like the alpha helix, all amide protons point toward the Nterminus (down) and all carbonyl oxygens point toward the C-terminus (up). Hydrogen bonds (i, i+3) are displayed as yellow line segments connecting the amide nitrogen and carbonyl oxygen pairs. The less than perfect alignment of these groups is apparent from the angle of the lines. Looking at the 3.10 helix along the helical axis from the C-terminus (top), you can see the three carbonyl oxygens of the last turn of the helix and the alignment of sidechains. 15.2.3 pi-helix The pi helix is an extremely rare secondary structural element in proteins. Hydrogen bonds within a pi-helix display a repeating pattern in which the backbone C=O of residue i hydrogen bonds to the backbone HN of residue i+5. Like the 3.10 helix, one turn of pi helix is sometimes found at the ends of regular alpha helices but pi helices longer than a few i, i+5 hydrogen bonds are not found. The infrequency of this particular form of secondary structure stems from the following properties: the phi and psi angles of the pure pi helix (-57.1, -69.7) lie at the very edge of an allowed, minimum energy region of the Ramachandran (phi, psi) map. the pi helix requires that the angle tau (N-CA-C') be larger (114.9) than the standard tetrahedral angle of 109.5 degrees. the large radius of the pi helix means the polypeptide backbone is no longer in van der Waals contact across the helical axis forming an axial hole too small for solvent water to fill. side chains are more staggered than the ideal 3.10 helix but not as well as the alpha helix. A good look at a geometrically pure pi helix is afforded by the CPK representation shown in Figure 76. Again notice how, like the alpha and 3.10 helices, all amide protons point toward the N-terminus (down) and all carbonyl oxygens point toward the C-terminus (up). Looking at the pi helix down the helical axis from the C-terminus (top), you can see the four carbonyl oxygens of the last turn of the pi helix and the spread of the sidechains.
5.3 TIGHT TURNS

It is well known that helices and -sheets are the major stabilizing structures in proteins. Segments of the protein chain which are not helical or -sheet have been generally designated as random coil or irregular regions. These non repetitive motif elements include tight turns, bulges, and random coil structures ( Richardson, 1981). There are different types of tight turns ( Chou 2000) depending upon the number of atoms forming the turn. These are as follows: Delta-turn - It is the smallest tight turn which involves only two amino acid residues and the intraturn hydrogen bond for a delta-turn is formed between the backbone NH(i) and the backbone CO(i+1).
Gamma-turn - It involves three amino acid residues and the intraturn hydrogen bond for a gamma-turn is formed between the backbone CO(i) and the backbone NH(i+2). Beta-turn - A beta-turn involves four amino acid residues and may or may not be stabilized by the intraturn hydrogen bond between the backbone CO(i) and the backbone NH(i+3). Alpha-turn - An alpha-turn involves five amino acid residues where the distance between the Calpha(i) and the Calpha(i+4) is less than 7 and the pentapeptide chain is not in a helical conformation. Pi-turn - It is the largest tight turn which involves six amino acid residues.
15.3.1 Gamma Turns Of tight turns, beta-turn and gamma-turns have received much attention and been studied in detail. The second most characterized turn class, after the beta turns, is the gamma turns. A gamma turn resembles a beta turn, but it has only three turn residues. A gamma turn is defined by the existence of a hydrogen bond between the CO group of i th residue and the NH of the (i+2)th residue.Earlier studies have shown that the gamma-turn structure is fairly common in proteins. The first example of a gamma-turn in a protein was described by Matthews (1972) at the end of a beta- hairpin in thermolysin. Most of the classic gamma-turns occur at the end of beta-hairpins, but very few of the inverse ( Sibanda and Thornton, 1985). Also, gamma turns occur at ligand binding sites or active sites, for example the loop where that catalytically important aspartate is located in serine proteases ( MilnerWhite, 1990). It has however been postualted that inverse gamma turns may function as intermediates in folding and thus stabilizing beta-strands before they become beta-sheets ( Milner-White, 1990). Recently, gamma-turns have brought attention through studies that describe the incorporation of peptide secondary structure mimetics into small bioactive peptides in the development of stable, effective and selective receptor ligands ( Alkorta et al., 1996).
Types of Gamma Turns

Gamma-turns are divided into two classes called inverse and classic ( Bystrov et al., 1969). They differ in that the main-chain atoms of the two forms are related by mirror symmetry (just as type I and I' or type II and II' beta-turns are). Of the two, classic ones are far less common, and those that do exist are frequently found at the loop ends of beta-hairpins. On the other hand inverse gamma-turns tend not to give rise to polypeptide chain reversal. The following figure shows the two types of gamma-turns:
Fig 66. Types of gamma turns
The dihedral angle intervals for classic and inverse gamma-turns are:
Table 6:
15.3.2 -turn
A -turn is a region of the protein involving four consecutive residues where the polypeptide chain folds back on itself by nearly 180 degrees ( Lewis et al. 1971, 1973; Kuntz,1972; Crawford et al.,1973; Chou & Fasman ,1974). It is these chain reversals which give a protein its globularity rather than linearity. The -turn was originally identified, in model building studies, by Venkatachalam (1968). He proposed three distinct conformations based on phi, psi values (designated I, II and III) along with their related turns (mirror images) which have the phi, psi signs reversed (I', II and III'), each of which could form a hydrogen bond between the main chain C=O (i) and the N-H (i+3). Subsequently, Lewis et al. (1973) examined the growing number of three-dimensional protein structures and suggested a more general definition of a -turn. This stated that the distance between the C alpha (i) and the C alpha(i+3) was <7 and the residues involved were not helical. They found that 25% of their extended -turns did not possess the intraturn hydrogen bond suggested by Venkatachalam. To include the new data they extended the classification of -turns to 10 distinct types (I,I',II,II',III,III',IV,V,VI and VII). These classes were defined not only by phi,psi angles, but also less stringent criteria. Richardson (1981) has since reappraised the situation, and has suggested that there are only 6 distinct types (I, I, II, II, VIa and VIb) based on phi, psi ranges, along with a miscellaneous category IV. The Richardson classification is the system most widely used at present. Two main types of -turns are Type-I and Type-II with their mirror images I' and II'.
Fig 67. Two types of -turns (Type-I and Type-II)
A -turn consists of four consecutive residues defined by positions i, i+1, i+2, i+3 which are not present in alpha- helix; the distance between Calpha(i) and Calpha(i+3) is less than 7 ( Richardson 1981; Rose et al. 1985) and the turn leads to reversal in the protein chain. -turns may or may not be accompained by the NH (i+3)-CO (i) hydrogen bond connecting the main chain atoms; CO of the ith residue and NH of (i+3)rd residue in the turn ( Lewis et al 1973; Nemethy and Scheraga 1980). Turns play an important role in globular proteins from both structural and functional points of view. A polypeptide chain cannot fold into a compact structure without the component of turns. Also, turns usually occur on the exposed surface of proteins and hence probably represent antigenic sites or involve molecular recognition. Thus, owing to the above reasons, the prediction of -turns in proteins is an important element of secondary structure prediction.
15.4 LET US SUM UP

The alpha helix is the most abundant helical conformation found in globular proteins Alpha helices sometimes begin or end with a single turn of a 3.10 helix (one hydrogen bond) although this accounts for <25% of residues with this classification. The phi and psi angles of the alpha helix (lie in the center of an allowed, minimum energy region of the Ramachandran (phi, psi) map.
A gamma turn is defined by the existence of a hydrogen bond between the CO group of
i th residue and the NH of the (i+2)th residue

Elaborate on a-helices and b-turns.

Give your views on different helices and analyze each. (Refer 5.2).
15.7 REFERENCES
Carl Branden and John Tooze. 1999. Introduction to Protein Structure 2nd ed. Garland Publishing: New York, NY. "Polypeptide chain configurations in crystalline proteins". Proceedings of the Royal Society A 203: 321. "The structure of fibrous proteins". Chemical Reviews 32: 195218.
Bragg, WL; Kendrew JC, Perutz MF (1950)
Huggins, M (1943)
LESSON-16 PROTEIN PROTEIN INTERACTIONS

Contents 16.0 Aims and objectives 16.1 Introduction 16.2Methods to investigate protein-protein interactions 16.2.3 Pull-down assays 16.2.4 Screening or confirmation of protein interactions: 16.2.5 Two-hybrid screening 16.2.6 In-vivo crosslinking of protein complexes 16.2.7 Tandem affinity purification (TAP) 16.2.8 Chemical crosslinking 16.2.9Quantitative immunoprecipitation
(QUICK)
combined
with
knock-down
16.2.10 Dual Polarisation Interferometry (DPI) 16.2.11 Static Light Scattering (SLS) 16.3 Protein-protein interaction network visualization 16.4 Protein-protein interaction prediction 16.4.1 Phylogenetic profiling 16.4.2 Identification of homologous interacting pairs 16.4.3 Identification of structural patterns 16.4.4 Bayesian network modelling 16.4.5 Relationship to docking methods 16.5 Let us sum up 16.6 Lesson End Activities 16.7 Check your Progress 16.8 References

In this unit, we have discussed about protein-protein interactions , methods to investigate Protein-protein interaction and their prediction methods
After going through this lesson , you must be able to : Explain methods to investigate protein-protein interactions Give account on protein-protein interaction prediction methods
16.1 INTRODUCTION:
Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks. The interactions between proteins are important for many biological functions. For example, signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signalling molecules. This process, called signal transduction, plays a fundamental role in many biological processes and in many diseases (e.g. cancer). Proteins might interact for a long time to form part of a protein complex, a protein may be carrying another protein (for example, from cytoplasm to nucleus or vice versa in the case of the nuclear pore importins), or a protein may interact briefly with another protein just to modify it (for example, a protein kinase will add a phosphate to a target protein). This modification of proteins can itself change protein-protein interactions. For example, some proteins with SH2 domains only bind to other proteins when they are phosphorylated on the amino acid tyrosine. In conclusion, protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches.
16.2 METHODS TO INVESTIGATE PROTEIN-PROTEIN INTERACTIONS

As protein-protein interactions are so important there are a multitude of methods to detect them. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur in reality are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are also occurring in reality. 16.2.1 Immunoprecipitation Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity. Co-immunoprecipitation (also known as a pull-down) can identify interacting proteins or protein complexes present in cell extracts: by precipitating one protein believed to be in a complex, additional members of the complex are captured as well and can be identified. The protein complexes, once bound to the specific antibody, are removed from the bulk solution by capture with an antibodybinding protein attached to a solid support such as an agarose bead. These antibodybinding proteins (Protein A, Protein G, Protein L) were initially isolated from bacteria and recognize a wide variety of antibodies. Following the initial capture of a protein or protein complex, the solid support is washed several times to remove any proteins not
specifically and tightly bound through the antibody. After washing, the precipitated protein(s) are eluted and analyzed using gel electrophoresis, mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex. Thus, coimmunoprecipitation is a standard method to assess protein-protein interaction. Immunoprecipitation is very useful in chromatin immunoprecipitation, which allows analysis of DNA sequences bound to histones or other chromatin components. Steps a) Lyse cells and prepare sample for immunoprecipitation. (you can radiolabel cells to easily assay for your protein later) b) Pre-clearing the sample using serum from the animal species in which the primary antibody was raised (this decreases background and non-specific proteins). c) Incubate solution with antibody against protein of interest (antibody can be attached to solid support before or after this step) and allow antibody-antigen complex formation. d) Precipitate the complex of interest, removing it from bulk solution. e) Wash precipitated complex several times. Spin each time between washes and remove the supernatant. After the last wash, get rid of as much supernatant as possible. f) Elute proteins from solid support (usually with low-pH or SDS sample loading buffer). g) Analyze complexes or antigens of interest. This can be done in a variety of ways: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); expose the film if you used radioactivity. Transfer and Western Blot using another antibody for proteins that were interacting with the antigen. 16.2.2 Fluorescence resonance energy transfer (FRET) It is a common technique when observing the interactions of only two different proteins. Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. FRET microscopy method can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide- field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled
with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein. 16.2.3 Pull-down assays Pull-down assays are a common variation of immunoprecipitation and are used identically. A pull-down assay is distinct from immunoprecipitation in that it uses some ligand other than an antibody to capture the protein complex. Discovery and confirmation of protein:protein interactions using the pull-down technique depend heavily on the nature of the interaction under study. Interactions can be stable or transient and this characteristic determines the conditions for optimizing binding between bait and prey proteins. Stable interactions make up most cellular structural features but can also occur in enzymatic complexes that form identifiable structures. Transient interactions are usually associated with transport or enzymatic mechanisms. The ribosome illustrates both examples because the structure consists of many stable protein:protein interactions, but the enzymatic mechanism that translates mRNA to nascent protein requires transient interactions. Stable protein:protein interactions are easiest to isolate by physical methods like pulldown assays because the protein complex does not disassemble over time. Since these interactions often contribute to cellular structure, the dissociation constant between proteins is usually low, correlating to a strong interaction. Strong, stable protein complexes can be washed extensively with high ionic strength buffers to eliminate any false positive results due to nonspecific interactions. If the complex interaction has a higher dissociation constant and is a weaker interaction, the interaction strength and thus protein complex recovery can be improved by optimizing the assay conditions related to pH, salt species and salt concentration. Problems of nonspecific interactions can be minimized with careful design of appropriate control experiments. Transient interactions are defined by their temporal interaction with other proteins and are the most challenging protein:protein interactions to isolate. These interactions are more difficult to identify using physical methods like pull-down assays because the complex may dissociate during the assay. Since transient interactions occur during transport or as part of enzymatic processes, they often require cofactors and energy via nucleotide triphosphates hydrolysis. Incorporating cofactors and nonhydrolyzable NTP analogs during assay optimization can serve to trap interacting proteins in different stages of a functional complex that is dependent on the cofactor or NTP. 16.2.4 screening or confirmation of protein interactions: Label transfer can be used for screening or confirmation of protein interactions and can provide information about the interface where the interaction takes place. Label transfer
can also detect weak or transient interactions that are difficult to capture using other in vitro detection strategies. In a label transfer reaction, a known protein is tagged with a detectable label. The label is then passed to an interacting protein, which can then be identified by the presence of the label. 16.2.5 Two-hybrid screening The yeast two-hybrid screen investigates the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify binding partners of a protein in an unbiased manner. However, the method has a notorious high false-positive rate which makes it necessary to verify the identified interactions by coimmunoprecipitation. Binding and activating domains of transcription factor GAL4 are split and fused to two proteins being tested for interactions. If the two proteins interact, transcription of "reporter gene" occurs. Otherwise, "reporter gene" is silent.
Fig 68 :Overview of two-hybrid assay
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For the purposes of two-hybrid screening, the transcription factor is split into two separate fragments, called the binding domain (BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for activation of transcription. 16.2.6 In-vivo crosslinking of protein complexes In-vivo crosslinking of protein complexes using photo-reactive amino acid analogs was introduced in 2005 by researchers from the Max Planck Institute. In this method, cells are grown with photoreactive diazirine analogs to leucine a n d methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog. 16.2.7 Tandem affinity purification (TAP) It detects interactions within the correct cellular environment (e.g. in the cytosol of a mammalian cell) (Rigaut et al., 1999). The Tandem Affinity Purification (TAP) method
involves the fusion of the TAP tag to the C-terminus of the new protein. The TAP tag consists of calmodulin binding peptide (CBP) from the N-terminal, followed by tobacco etch virus protease (TEV protease) cleavage site and Protein A, which binds tightly to IgG. The relative order of the modules of the tag is important because Protein A needs to be at the extreme end of the fusion protein so that the entire complex can be retrieved using an IgG matrix. There are a few methods in which the fusion protein can be introduced into the host. If the host is yeast, then one of the methods may be the use of plasmids that will eventually translate the fusion protein within the host. Whichever method that is being used, it is preferable to maintain expression of the fusion protein as close as possible to its natural level. Once the fusion protein is transcribed within the host, the new protein at one of the fusion protein would be able to interact with other proteins. Subsequently, the fusion protein is retrieved from the host by breaking the cells and retrieving the fusion protein through affinity s e lection, together with the other constituents attached to the new protein, by means of an IgG matrix. After washing, TEV protease is introduced to elute the bound material at the TEV protease cleavage site. This eluate is then incubated with calmodulincoated beads in the presence of calcium. This second affinity step is required to remove the TEV protease as well as traces of contaminants remaining after the first affinity step. After washing, the eluate is then released with ethylene glycol tetraacetic acid (EGTA). The native elution consisting of the new protein, its interacting protein partners as well as CBP can now be analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or be identified by mass spectrometry. This is a big advantage compared to the yeast two- hybrid approach. However, there is also the possibility that a tag added to a protein might obscure binding of the new protein to its interacting partners. In addition, the tag may also affect protein expression levels. On the other hand, the tag may also not be sufficiently exposed to the affinity beads, hence skewing the results.There may also be a possibility of a cleavage of the proteins by the TEV protease, although this is unlikely to be frequent given the high specificity of the TEV protease.Thus, it can not readily detect transient protein-protein interactions. 16.2.8 Chemical crosslinking Chemical crosslinking is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. A variety of crosslinkers are used to analyze proteinprotein interactions and various parameters of protein function. Subunit structure is deduced since crosslinkers only bind surface amino residues in relatively close proximity in the native state. Protein interactions are often too weak or transient to be easily detected, but by crosslinking, the interactions can be captured and analyzed. Examples of some common crosslinkers are the imidoester crosslinker dimethyl suberimidate, the NHS-ester c r o s slinker BS3 and formaldehyde. Each of these crosslinkers induces nucleophilic attack of the amino group of lysine and subsequent covalent bonding via the crosslinker. The zero- length carbodiimide crosslinker EDC functions by converting caboxyls into amine-reactive isourea intermediates that bind to lysine residues or other available primary amines.
In-vivo crosslinking of protein complexes using photo-reactive amino acid analogs was introduced in 2005 by researchers from the Max Planck Institute. In this method, cells are grown with photoreactive diazirine analogs to leucine a n d methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog. 16.2.9 Quantitative immunoprecipitation combined with knock-down (QUICK) It relies on coimmunoprecipitation, quantitative mass spectrometry (SILAC) and RNA interference (RNAi). This method detects interactions among endogenous non-tagged proteins (Selbach and Mann, 2006). Thus, it has the same high confidence as coimmunoprecipitation. However, this method also depends on the availability of suitable antibodies. 16.2.10 Dual Polarisation Interferometry (DPI) This can be used to measure protein-protein interactions. DPI provides real- time, highresolution measurements of molecular size, density and mass. While tagging is not necessary, one of the protein species must be immobilized on the surface of a waveguide. 16.2.11 Static Light Scattering (SLS) Static Light Scattering is a sensitive and precise measure of weight-averaged molar mass that can non-destructively characterize both weak and strong interactions without tagging or immobilization of the protein. The measurement consists of introducing a series of aliquots of different concentrations and/or compositions into the detector, and fitting the resultant signals to equations representing the effect of the interaction on the light scattering. Weak, non-specific interactions are typically characterized via the second virial coefficient. Stronger interactions with specific stoichiometries, manifesting as reversible homo- and hetero-associations, are analyzed by fitting the signals to association models. The analysis determines the equilibrium association constant for each associated complex present in the solution. Association kinetics may also be characterized via SLS. A system incorporating a static light scattering detector, an on- line concentration detector, and automation of the sample preparation, delivery, and data acquisition, as well as analysis of virial coefficients and reversible protein associations. Chemical crosslinking followed by high mass MALDI mass spectrometry can be used to analyze intact protein interactions in place before trying to isolate/identify interacting proteins. This method detects interactions among non-tagged proteins and is available from CovalX.
16.3 PROTEIN-PROTEIN INTERACTION NETWORK VISUALIZATION

Visualization of protein-protein interaction networks is a popular application of scientific visualization techniques. Although protein interaction diagrams are common in
textbooks, diagrams of whole cell protein interaction networks were not as common since the level of complexity made them difficult to generate. One example of a manually produced molecular interaction map is Kurt Kohn's 1999 map of cell cycle control. Drawing on Kohn's map, in 2000 Schwikowski, Uetz, and Fields published a paper on protein-protein interactions in yeast, linking together 1,548 interacting proteins determined by two-hybrid testing. They used a force-directed (Sugiyama) graph drawing algorithm to automatically generate an image of their network. Protein-protein interaction prediction
16.4 PROTEIN-PROTEIN INTERACTION PREDICTION

It is a field combining bioinformatics and structural biology in an attempt to identify and catalog interactions between pairs or groups of proteins. Understanding protein-protein interactions is important in investigating intracellular signaling pathways. Experimentally, interactions between pairs of proteins are inferred from yeast two-hybrid systems, from affinity purification/mass spectrometry assays, or from protein microarrays. In parallel to the experimental determination of the interactome, computational methods are being developed. Methods Proteins that interact are more likely to co-evolve, therefore it is possible to make inferences about interactions between pairs of proteins based on their phylogenetic distances. It has also been observed in some cases that pairs of interacting proteins have fused orthologues in other organisms. In addition, a number of bound protein complexes have been structurally solved and can be used to identify the residues that mediate the interaction so that similar motifs can be located in other organisms. 16.4.1 Phylogenetic profiling This method involves using a sequence search tool such as BLAST for finding homologues of a pair of proteins, then building multiple sequence alignments with alignment tools such as Clustal. From these multiple sequence alignments, phylogenetic distance matrices are calculated for each protein in the hypothesized interacting pair. If the matrices are sufficiently similar (as measured by their Pearson correlation coefficient) they are deemed likely to interact. 16.4.2 Identification of homologous interacting pairs This method consists of searching whether the two sequences have homologues which form a complex in a database of known structures of complexes. The identification of the domains is done by sequence searches against domain databases such as Pfam using BLAST. If more than one complex of Pfam domains is identified, then the query sequences are aligned using a hidden Markov tool called HMMer to the closest identified homologues, whose structures are known. Then the alignments are analysed to check whether the contact residues of the known complex are conserved in the alignment.
16.4.3 Identification of structural patterns A third method builds a library of known protein-protein interfaces from the PDB, where the interfaces are defined as pairs of polypeptide fragments that are below a threshold slightly larger than the Van der Waals radius of the atoms involved. The sequences in the library are then clustered based on structural alignment and redundant sequences are eliminated. The residues that have a high (generally >50%) level of frequency for a given position are considered hotspots. This library is then used to identify potential interactions between pairs of targets, providing that they have a known structure (i.e. present in the PDB). 16.4.4 Bayesian network modelling Bayesian methods integrate data from a wide variety of sources, including both experimental results and prior computational predictions, and use these features to assess the likelihood that a particular potential protein interaction is a true positive result. These methods are useful because experimental procedures, particularly the yeast two-hybrid experiments, are extremely noisy and produce many false positives, while the previously mentioned computational methods can only provide circumstantial evidence that a particular pair of proteins might interact. 16.4.5 Relationship to docking methods The field of protein-protein interaction prediction is closely related to the field of proteinprotein docking, which attempts to use geometric and steric considerations to fit two proteins of known structure into a bound complex. This is a useful mode of inquiry in cases where both proteins in the pair have known structures and are known (or at least strongly suspected) to interact, but since so many proteins do not have experimentally determined structures, sequence-based interaction prediction methods are especially useful in conjunction with experimental studies of an organism's interactome.
16.5 LET US SUM UP

Protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches. Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. Co-immunoprecipitation is a standard method to assess protein-protein interaction. Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A A pull-down assay is distinct from immunoprecipitation in that it uses some ligand other than an antibody to capture the protein complex
Interactions between pairs of proteins are inferred from yeast two-hybrid systems, from affinity purification/mass spectrometry assays, or from protein microarrays.

1. Elucidate the fact that protein-protein interactions are of central importance for every process in a living cell. 2. How important in the prediction of protein protein interaction?

What is the advantage of protein protein interaction visualization? (Refer 16.3).

(1) Erica Golemis Protein-Protein Interactions: A Molecular Cloning Manual
(2) http://www.functionalgenomics.org.uk/sections/resources/protein-protein.htm (3) http://www.biochem.ucl.ac.uk/bsm/PP/server/
LESSON 17 - PROTEIN-DNA INTERACTION

Contents 17.0 Aims and objectives 17.1 Introduction 17.2 Types of Protein-Nucleic Acid Interactions 17.3 In Vitro Methods for Protein: Nucleic Acid Interaction Analysis 17.4 Protein: DNA Crosslinking 17.4.1 Agents that Crosslink DNA 17.5 Affinity-based Methods 17.5.1 DNA Foot printing 17.6 Protein-DNA interaction site predictor 17.7 Let us sum up 17.8 Lesson End Activities 17.9 Check your Progress 17.10 References

In this unit we disscuss the nature of protein DNA interaction,types of protein DNA interaction and methods for Protein: Nucleic Acid Interaction Analysis. After going through this unit, you will be able to: List various methods usd for the investigation of protein-DNA analysis Explain types of Protein-Nucleic Acid Interactions Explain protein:DNA Cross linking
17.1 INTRODUCTION
Proteins and nucleic acids do not operate within the biological system as independent entities. Protein: nucleic acid interactions, (i.e., protein:RNA and protein:DNA interactions), are involved in several processes essential to normal cell function. As with protein: protein interactions, disruption of protein: nucleic acid interactions lead to serious and often catastrophic consequences within the system. Protein-Nucleic acid interactions are integrated into several key cellular processes. These processes include transcription, translation, and regulation of gene expression, recognition, replication, recombination, repair, nucleic acid packaging and the formation
of cellular machinery, such as ribosome. The role of DNA as the genetic repository of information requires interaction with proteins for the extraction of this information for timely utilization within the cell. 17.2 TYPES OF PROTEIN-NUCLEIC ACID INTERACTIONS The common property of nucleic acid binding proteins is their ability to recognize and manipulate DNA/RNA structures. Transcription complex formation, initiation of transcription, and translation of messenger RNA to protein all involve formation of protein: nucleic acid complexes containing either DNA or RNA. These complexes by their nature play a role in the regulation of protein expression. Depending on the nature of the complex, proteins bind to nucleic acids in either a sequence-specific or secondary structure-dependent manner, often inducing drastic structural changes in the nucleic acid. Proteins can interact with nucleic acids in a variety of modes involving either major or minor groove associations. Defining sequence-specific interactions can aid in the development of high affinity aptamers, which may be used as purification tools for DNA or RNA binding proteins. Sequence-specific interactions also have application in the study of gene regulation and drug discovery. 17.3 IN VITRO METHODS FOR PROTEIN: NUCLEIC ACID
INTERACTION ANALYSIS
These methods provide specific information as to the binding site locus of a DNA binding protein to a nucleic acid substrate. Electrophoretic Mobility Shift Assay (EMSA) The electrophoresis mobility shift assay (EMSA) is a rapid and sensitive method to detect proteinnucleic acid interactions. It is based on the observation that the electrophoretic mobility of a proteinnucleic acid complex is typically less than that of the free nucleic acid. The current, widely- used assay differs little from that originally described by Fried and Crothers and Garner and Revzin; although precursors to the technique can be found in the earlier literature. Mobility-shift assays are often used for qualitative purposes; although under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, affinities and kinetics. Methods used in performing the assay differ for each purpose, and a large number of variants have been described in the literature.
Fig 69. Functional mapping of in vitro DNA binding activity using EMSA with purified Igs protein from bacterial cell extracts
Advantages and limitations of EMSA The mobility shift assay has a number of strengths. The basic technique is simple to perform, yet it is robust enough to accommodate a wide range of binding conditions. Using radioisotope-labeled nucleic acids, the assay is highly sensitive, allowing the assays to be performed with small protein and nucleic acid concentrations (0.1 nM or less) and small (20 l or less) sample volumes. When such high sensitivity is not needed, variants or the assay using fluorescence, chemiluminescence and immunohistochemical detection are also available. A wide range of nucleic acid sizes (ranging from short oligonucleotides to several thousand nucleotides (nt) or base pairs (bp)) and structures (single-stranded, duplex, triplex and quadruplex nucleic acids as well as small circular DNAs) are compatible with the assay. Under favorable conditions, the distribution of proteins between several nucleic acid molecules can be monitored within a single solution, as can the presence of complexes differing in protein stoichiometry and/or binding site distribution. Proteins ranging in size from small oligopeptides to transcription complexes with Mr 106 can give useful mobility shifts and the assay works well with both highly purified proteins and crude cell extracts. These capabilities account in large part for the continuing popularity of the assay. On the other hand, the EMSA is not without limitations. One theme of this protocol is the identification of potential problems and the suggestion of strategies that avoid or mitigate the most severe. The TROUBLESHOOTING section contains a guide for troubleshooting the most common problems that the authors have encountered. Perhaps the most important limitation is that samples are not at chemical equilibrium during the electrophoresis step. Rapid dissociation during electrophoresis can prevent detection of complexes, while even slow dissociation can result in underestimation of binding density. On the other hand, many complexes are significantly more stable in the gel than they are
in free solution; when this is the case, short electrophoresis times allow the resolution of patterns that closely approximate the distributions of species present in the samples at the start of electrophoresis. A second limitation is that the electrophoretic mobility of a proteinnucleic acid complex depends on many factors other than the size of the protein. Thus, an observed mobility shift does not provide a straightforward measure of the molecular weights or identities of proteins that are present in the complex. The electrophoretic 'supershift' assay and assays that combine EMSA with western blotting or mass spectroscopy have been devised to allow identification of nucleic acid-associated proteins, while a range of EMSA-based and non-EMSA methods can be used for evaluation of binding stoichiometries. A third limitation is that the electrophoretic mobility of a complex provides little direct information about the location of the nucleic acid sequences that are occupied by protein. This information is available from nuclease and chemical footprinting assays that can be performed independently of EMSA or in concert with it. Finally, the time resolution of the current assay is defined by the interval required for manual solution handling. This limits kinetics studies to processes with relaxation times significantly larger than approximately 1 min required to mix reaction components and for electrophoretic migration into the gel matrix. Strategies designed to improve the time resolution of the technique are in development (M.G.F., unpublished results). Alternatives to EMSA Many techniques are available for the detection and characterization of proteinnucleic acid complexes and most have advantages and disadvantages that differ from those of the EMSA. The most widely used alternative assays are nitrocellulose filter-binding and footprinting. Filter binding is simple to perform and the manipulations are rapid enough to allow kinetic studies as well as equilibrium measurements. Under favorable conditions, the assay can be highly sensitive and with the exception of the scintillation counter, the required equipment is inexpensive. Like the EMSA, filter-binding is a nonequilibrium technique. As a result, quantitative analyses require careful evaluation of filter-retention efficiency. The assay is not limited by the salt concentration of the sample and it can accommodate very large nucleic acids (e.g., the phage genome (48,502 bp)). In contrast, the electrophoretic process limits the salt concentrations of EMSA samples to 300 mM or less (1:1 salt) and to DNAs of 5,000 bp or less (note that these limits are approximate and may vary with sample and gel compositions). The presence of more than one binding protein complicates filter-binding analysis, since retention of labeled nucleic acid is detected, and not the identity of bound proteins or the proportion of binding activity attributable to each. In addition, conditions that give efficient filter-retention of 1:1 proteinnucleic acid complexes do not result in significantly increased retention of higher stoichiometry complexes, hence the classical assay is poorly suited for distinguishing complexes containing one protein from those containing more than one. Finally, under some solution conditions, single-stranded nucleic acids are retained by nitrocellulose filters, resulting in a background of retained radioactivity that can mask the binding signal. Thus, for applications involving more than one nucleic acid-binding protein, the
detection of more than one proteinnucleic acid complex in a mixture and/or binding to single-stranded nucleic acids, mobility shift assays offer clear advantages. Footprinting assays exploit the observation that a protein bound to a specific nucleic acid sequence will interfere with the chemical or enzymatic modification of that sequence. A large number of chemical and enzymatic agents are available for this purpose. In the classical assay, a radioisotope label is located at one end of one strand of the nucleic acid target. Following modification and any steps needed to cleave the nucleic acid at modification sites, fragments are resolved on a denaturing polyacrylamide gel. The resulting ladder of bands is visualized by autoradiography and gaps in the array indicate sites of protection by the protein(s) in the test mixture. Comparison of the protection pattern with sequencing reaction products can allow identification of protected sequences with single-nucleotide resolution. In addition to protection, the appearance of sites that are hypersensitive to modification can provide evidence of conformational change in the target nucleic acid. Because most other methods provide little direct information about the identities of protein binding sites, this method remains the 'gold standard' for the identification of nucleic acid sequences within or near binding sites. Since sequences of several hundred residues can be resolved on a typical sequencing gel, the technique is well suited for the analysis of the binding of several protein molecules to a single nucleic acid (a strength that footprinting shares with EMSA). In addition, the footprint signal can be obtained under conditions of binding equilibrium for the protein of interest. This is an important advantage over nonequilibrium assays such as EMSA. Variants of the footprinting assay optimized for quantitative detection of binding have been described, and time-resolved methods have been developed that allow the analysis of binding kinetics as well as equilibria. Footprinting assays require simultaneous optimization of binding by the protein(s) of interest and the nucleic acid modification reaction(s) needed to produce the footprint signal. Thus, they are somewhat more difficult to perform than EMSA or filter binding assays. In addition, because the radioisotope label is distributed over many nucleic acid fragments, the detection of binding by footprinting is less sensitive than the detection of binding using EMSA. In addition, incomplete binding results in a footprint pattern that contains contributions from both free and bound nucleic acids. This makes the protected regions indistinct, because the pattern of free nucleic acid is visible within the protected footprint. A popular solution to this problem is to use EMSA to separate complexes from free nucleic acids after the footprinting modification reaction. Following this step with denaturing gel, electrophoresis allows one to independently visualize the fragment patterns of free and bound nucleic acid populations. Finally, some proteins that bind nucleic acids without sequence specificity may not produce distinct footprints. For such systems, EMSA and filter binding assays provide binding signals that are easier to interpret than those available from footprinting. Strategic considerations that are relevant to EMSA No single set of binding and electrophoresis conditions works well for all molecular systems. However, several variables can be optimized for the study of a particular
interaction, including design of the nucleic acid target, binding reaction conditions and electrophoresis conditions, as discussed in the following text. Selection of nucleic acid target. Short nucleic acids are easily synthesized and inexpensive to purchase. The small number of nonspecific protein binding sites in a small DNA or RNA can be advantageous when the binding protein has low sequence-specificity. In addition, electrophoretic resolution of complexes from free nucleic acid is highest with small nucleic acids and this makes short electrophoresis times possible. On the other hand, all binding sites on a short nucleic acid are close to the molecular ends. This can result in aberrant binding due to structural and electrostatic end-effects. Longer templates avoid these limitations but contain more nonspecific binding sites, migrate more slowly (requiring longer electrophoresis times) and generally give a smaller mobility-shift on protein binding. In the classical EMSA, the electrophoretic mobility of the nucleic acid is monitored. Nucleic acids can be labeled with radioisotopes, covalent or noncovalent fluorophores or biotin. These labels can be detected by autoradiography, fluorescence imaging, chemiluminescent imaging and/or chromophore deposition, respectively. Alternatively, unlabeled nucleic acid can be used in the binding and electrophoresis steps and detected by postelectrophoretic staining with chromophores or fluorophores that bind nucleic acids. Labeling the nucleic acid at the 5'- or 3'-end with [32 P]phosphate is a widely used approach, as it is inexpensive, offers great sensitivity (10-18 mol or less of 5'-ends can be detected on a routine basis) and does not introduce artificial structures that might influence binding. Thus, when radioisotope use is permissible, we consider 32 P-labeling the method of choice for making nucleic acids detectable in the EMSA. When radioisotope use is not possible, covalent labeling of nucleic acid with a fluorophore allows both qualitative and quantitative EMSA applications, although with lower sensitivity than is possible with radioisotope- labeled nucleic acids. Binding conditions. Proteinnucleic acid interactions are sensitive to mono- and divalent salt concentrations and pH. Although a typical Tris-based sample buffer is used in the protocol detailed here, good results have been obtained with many different buffers, including HEPES, MOPS (3-(N-morpholino)propanesulfonic acid), Bis-Tris (1,3-Bis (tris (hydroxymethyl) methylamino) propane), Gly and phosphate. We favor buffers that approximate physiological salt concentrations and pH and provide any needed cofactors at appropriate concentrations. However, as long as the conductivity of the sample is not excessive, electrophoresis can be carried out with a wide range of sample compositions. This allows the concentration of binding buffer components to be adjusted to optimize complex formation. Additives. Small neutral solutes such as glycerol or sucrose are often used to stabilize labile proteins. Such solutes can also enhance the stabilities of proteinnucleic acid interactions and can be valuable additions to binding reaction mixtures. They are generally effective when the solute concentration is less than 2 M, but less is known about their effects at higher concentrations, where high viscosity and surface tension complicate solution
handling. Addition of modest concentrations (0.1 mg ml-1 or less) of a carrier protein (e.g., BSA) to the binding reaction can minimize nonspecific losses of binding proteins during solution handling. Similarly, nonionic detergents are sometimes helpful in maximizing protein solubility. The useful concentrations of detergents vary with the identities of the detergent and the molecular system under study. Protease, nuclease and phosphatase inhibitors can be useful additives, especially when the protein sample is a partially- fractionated cell or nuclear extract. Mixtures of inhibitors are commercially available and should be used according to the supplier's instructions. Finally, some systems require specific cofactors for correct function. Examples include cAMP for the Escherichia coli CAP (cAMP receptor) protein, ATP for recombinases such as E. coli RecA or human Rad51 and polyamines for some eukaryotic transcription factors. Where necessary, smallmolecule additives that stabilize complexes can be included in binding and gel buffers, to stabilize complexes during electrophoresis. Competing nucleic acid. Often a protein sample will contain more than one nucleic acid binding activity. When secondary binding activities obscure the one of interest, the addition of unlabeled competing nucleic acid to the reaction mixture can reduce the binding of secondary proteins to the labeled target. This strategy works when the protein of interest binds the target nucleic acid with greater affinity than it binds the competitor and when the secondary binding activities do not discriminate between competitor and target sequences. Since competing nucleic acids also reduce the amount of specific binding, even under favorable conditions, it is best to test a range of competitor concentrations to optimize discrimination of specific and nonspecific binding. Commonly used competitors include genomic DNAs, poly d(AT) and poly d(IC). Electrophoresis conditions. The resolution of complexes depends on their stability during electrophoresis. Since many buffers are compatible with electrophoresis, the composition and concentration of gel and running buffer components can be adjusted to optimize the stability of complexes. The most popular buffers are variants of TrisborateEDTA, Trisacetate EDTA or TrisGly. As previously mentioned, low molecular weight cofactors and/or nonspecific stabilizers such as glycerol or ethylene glycol can be included in gel buffers and running buffers to enhance the stability of complexes. In some circumstances, it may be feasible to perform electrophoresis in the buffer that was used in the binding reaction. This avoids subjecting the sample to a change of buffer conditions during gel loading and as reaction components migrate into the gel. This has the advantage of avoiding any perturbation to the molecular system that might be due to a change in buffer composition; it also eliminates the need to independently optimize binding and electrophoresis buffer conditions. Although both polyacrylamide and agarose gels have been used for EMSA, polyacrylamide gels offer better electrophoretic resolution for proteinDNA and protein RNA complexes of Mr 500,000. In addition, some complexes are significantly more stable in polyacrylamide matrices than in agarose gels or free solution. While the mechanism of this effect is not completely understood, for a few molecular systems it has
been shown that the dissociation rates of complexes decrease with increasing gel concentration. On the other hand, the resolution of large complexes and the duration of electrophoresis is much longer in high percentage gels. Thus, optimization of gel concentration can be a valuable precursor to the most critical EMSA applications. One effective optimization strategy is to start with a relatively low concentration gel (e.g., 5% wt/vol acrylamide) and to increase this concentration systematically until any improvement in complex stability is balanced by loss of electrophoretic resolution and/or the onset of impractically long electrophoresis times.
17.4 PROTEIN: DNA CROSSLINKING

Crosslinks in DNA occur when various exogenous or endogenous agent react with two different positions in the DNA. This can either occur in the same strand (intrastrand crosslink) or in the opposite strands of the DNA (interstrand crosslink). Crosslinks also occur between DNA and protein. DNA replication is blocked by crosslinks and leads to cell death. 17.4.1 Agents that Crosslink DNA I. Exogenous Cross Linking Agents Alkylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU, Carmustine) and Nitrogen mustard which are used in chemotherapy can cross link with DNA at N7 position of guanine on the opposite strands forming interstrand crosslink. Cisplatin (cis-diamminedichloroplatinum(II) and its derivative forms DNA cross links as monoadduct, interstrand crosslink, intrastrand crosslink or DNA protein crosslink. Mostly it acts on the adjacent N-7 guanine forming 1, 2 intrastrand crosslink. II. Endogenous Cross Linking Agents a) Nitrous acid formed in the stomach dietary source nitrites. It induces formation of interstrand DNA crosslink at aminogroup of exocyclic N2 of guanine at the CG sequences. b) Reactive chemicals such as malondialdehyde which are formed endogenously as the product of lipid peroxidation. They create etheno adducts formed by aldehyde which undergo rearrangements to form crosslinks on opposite strands. III Psoralens: Psoralens are natural compounds (furocoumarins) present in plants. These compounds get activated in the presence of UV - A. They form covalent adducts with pyrimidines. Covalent adducts are formed by linking 3, 4 (pyrone) or 4', 5 (furan) edge of psoralen to 5, 6 double bond of thymine. Psoralens can form two types of monoadducts and one diadduct (an interstrand crosslink) reacting with thymine [5]. The crosslinking reaction by Psoralens targets TA sequences intercalating in DNA and linking one base of the DNA with the one below it. Psoralen adducts cause replication arrest and is used in the treatment of psoriasis and vitiligo.
DNA Protein Crosslink Aldehydes such as acrolein and crotonaldehyde found in tobacco smoke or automotive exhaust can form DNA interstrand crosslinks in DNA. Guanine adducts of DNA can also react with protein. A Schiff base formation between protein and aldehyde causes this DNA protein interstrand link Formaldehyde (HCHO) induces protein-DNA and protein-protein crosslinks, and is a common reagent of choice for molecular biology experiments.[6] These crosslinks may be reversed by incubation at 70C.
17.5 AFFINITY-BASED METHODS

Uses labeled DNA or RNA fragments bound to an affinity support to capture or purify specific binding proteins from crude extracts 17.5.1 DNA Footprinting A DNase footprinting assay is a technique from molecular biology that detects DNAprotein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on cellular DNA. The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end- labelled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern. For example, the DNA fragment of interest may be PCR amplified using a 32 P 5' labelled primer, with the end result being many DNA molecules with a radioactive label on one end of one strand of each double stranded molecule. Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel. The fragments which are smaller with respect to the 32 P-labelled end will appear further on the gel than the longer fragments. The gel is then used to expose a special photographic film. The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint". By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed. This technique was developed by David Galas and Albert Schmitz at Geneva on 1977
Fig 70 : The protein protects the binding site region from cleavage by DNase or another treatment.
17.6 PROTEIN-DNA INTERACTION SITE PREDICTOR

Structural and physical properties of DNA provide important constraints on the binding sites formed on surfaces of DNA-binding proteins. Characteristics of such binding sites may be used for predicting DNA-binding sites from the structural and even sequence properties of unbound proteins. This approach has been successfully implemented for predicting the protein-protein interface. Here, this approach is adopted for predicting DNA-binding sites in DNA-binding proteins. First attempt to use sequence and evolutinary features to predict DNA-binding sites in proteins were made by Ahmad et al. (2004) and Ahmad and Sarai (2005). Some methods use structural information to predict DNA-binding sites and therefore require a 3-dimensional structure of the protein, while others use only sequence information and do not require protein structure in order to make a prediction. Structure- and sequence-based prediction of DNA-binding sites in DNA-binding proteins can be performed on several web servers. 1) DISPLAR makes a prediction based on properties of protein structure. Knowledge of the protein structure is required 2) BindN makes a prediction based on chemical properties of the input protein sequence. Knowledge of the protein structure is not required. 3) DP-Bind combines multiple methods to make a consensus prediction based on the profile of evolutionary conservation and properties of the input protein sequence. Profile of evolutionary conservation is automatically generated by the web-server. Knowledge of the protein structure is not required. 4) DBS-PSSM (This article also shows how prediction can be significantly sped up by generating alignments against limited data sets). 5) DBS-Pred (This artcile also uses amino acid composition analysis to predict DNAbinding proteins, and uses structure information to improve binding site prediction. Method is based on single sequences only and thousands of proteins can be processed in less than an hour). Standalone is also available.
17.7 LET US SUM UP

Disruption of protein: nucleic acid interactions lead to serious and often catastrophic consequences within the system EMSA is based on the observation that the electrophoretic mobility of a protein nucleic acid complex is typically less than that of the free nucleic acid. DNA replication is blocked by crosslinks and leads to cell death. DNA footpriting makes it possible to locate a protein binding site on cellular DNA.

Highlight the features of different types of protein nucleic acid interactions.

Write a note and review on DNA foot printing (Refer 17.5.1).
17.10 REFERENCES
(1) http://www.biologie.uni- hamburg.de/lehre/bza/eprotdna.htm (2) http://www.biochem.ucl.ac.uk/bsm/prot_dna/prot_dna_cover.html (3)http://www.biochem.arizona.edu/classes/bioc568/protein_dna_interactions.htm
LESSON 18 DNA- DRUG INTERACTIONS, METALLOPROTEINS AND PROTEIN LIGAND INTERACTIONS

Contents 18.0 Aims and objectives 18.1 Introduction 18.2 DNA - Drug Interaction 18.2.1 Modeling DNA-ligand interaction of intercalating ligands 18.2.2 Rational for drug design 18.2.3 Modeling DNA-ligand interaction of minor groove binders 18.2.4 Drugs that form covalent bonds with DNA targets 18.3 Metalloproteins 18.3.1 Examples 18.3.2 Significance of metal-protein interaction 18.4 Protein-ligand interaction 18.4.1 Ligand binding sites of immunoglobin: 18.4.2 Model Building of immunoglobin Ligand System 18.5 Let us sum up 18.6 Lesson End Activities 18.7 Check Your Progress 18.8 Suggested Readings/References/Sources

In this lesson we have described briefly about DNA- Drug Interactions, Metalloproteins and Protein - Ligand Interactions After going through this chapter, you must be able to: Explain the steps involved in DNA - Drug Interaction Explain the significance of metal-protein interaction Explain the protein- ligand interaction
18.1 INTRODUCTION
DNA as carrier of genetic information is a major target for drug interaction because of the ability to interfere with transcription (gene expression and protein synthesis) and DNA replication, a major step in cell growth and division. The latter is central for tumorigenesis and pathogenesis. There are three principally different ways of drug-binding. First, through control of transcription factors and polymerases. Here, the drugs interact with the proteins that bind to DNA. Second, through RNA binding to DNA double helices to form nucleic acid triple helical structures or RNA hybridization (sequence specific binding) to exposed DNA single strand regions forming DNA-RNA hybrids that may interfere with transcriptional activity. Third, small aromatic ligand molecules that bind to DNA double helical structures by (i) intercalating between stacked base pairs thereby distorting the DNA backbone conformation and interfering with DNA-protein interaction or (ii) the minor groove binders. The latter cause little distortion of the DNA backbone. Both work through non covalent interaction. The small ligand drug approach offers a simple solution. The synthesis and screening of synthetic compounds that do not exist in nature, work much like pharmacological ligand for cell surface receptors in excitable tissue, and appear to be more readily delivered to cellular targets than large RNA or protein ligands. The lack of sequence specificity for intercalating molecules, however, does not allow to target specific genes, but rather certain cellular states or physiological and pathological conditions, like rapid cell growth and division that can be selectively suppressed as compared to non growing or slowly growing healthy tissue. Proteins are selective in their interactions with cell constituents. In contrast to naturally occurring proteins, chemically synthesized polypeptides of random sequence behave like small children; they touch, bind and break many low molecular weight metabolites. Natural proteins were educated by evolution to touch only a small selection of molecules .This was only possible because they learned to form defined compact structures in contrast to synthetic polypeptides. Specific binding is an individual property of individual proteins. Nonbinding rather than binding, is the result of organized protein structures. Relatively few types of molecules play a role in living systems as cofactors or metabolites. Apart from ions typical examples are derivatives of adenosine, of glucose, of porphyrine, and of - amino acids. Consequently most proteins are specialized in interacting with one or more of such standard building blocks.
18.2 DNA - DRUG INTERACTION

18.2.1 Modeling DNA-ligand interaction of intercalating ligands
The following properties have been identified as important for the successful modeling of ligand-DNA interaction: - degrees of freedom, - role of base pair sequence, - counter ion effects , - role of solvent ligand-receptor binding. Degrees of freedom: This problem is analogous to that of protein ligand interaction. The major requirement for intercalating agents is the planar aromatic ring structure. This structure fits between to adjacent base pair planes and can have some, although much restricted, rotational freedom within the plane of the ring. The ligand itself may have flexibility of structural parts outside the DNA binding site and may contain more than one intercalating sidechain:
Fig 71 : The structure of the antibiotic triostin A
The structure of the antibiotic triostin A shows the presence of two quinoxaline (groups to the right; double aromatic rings) units linked through a cyclic peptide structure (center left) which is stabilized at its center by a cystein pair (disulfhydril covalent bond).
Fig 72. Chemical structure of triostatin A.
The space filled side view indicates how the two quinoxaline rings are positioned by the linker peptide in co-planar fashion suitable for intercalating with DNA base pairs. As a rule, the more intercalating side chains are linked within a single ligand structure, the stronger the expected binding affinity. Triostatin A belongs to a family of antibiotics which are characterized by crosslinked octapeptide rings bearing two quinoxaline chromophores. Since the spacing between the chromophores is 3.5A, the intercalation process sandwiches two base pairs between the
two quinoxalines. This phenomenon is called bis-intercalation and has first been described for echinomycin by showing that bis-intercalating drugs cause twice the DNA helix extension and unwinding seen as compared to single intercalating molecule like ethidium. The latter is a chromophore which is activated by UV light and is used by molecule biologists to label nucleic acids in gel electrophoresis or ion gradient centrifugation. Role of base pair sequence: Experimental evidence suggests that base pair sequence does not play a large role on the specific mature of most intercalating complexes. As the structure of triostatin A suggests, however, the linker peptide structure may well promote specific interaction with the DNA surface. The major group specific readout sequence of H-bond donor and acceptor could be involved in triostatin A binding. The figure graphically shows the direct readout of the DNA base sequence on a double helical structure.
Fig 73. Overlap of AT and GC base pairs
The following characteristics of non covalent bond formation are associated with the binding sites indicated above: binding site W1 W2 W3 W3' W2' W1' GC base pair H-bond acceptor blank H-bond acceptor blank H-bond donor C-H weak hydrophobic
Table 7:
AT base pair H-bond acceptor blank H-bond donor blank H-bond acceptor CH3, strong hydrophobic
While the interaction on the major groove side is distinct for the direction of the base pair (e.g. AT vs TA), there is no directionality at the minor groove side. The molecular basis of specific recognition between echinomycin and DNA is due to the hydrogen bonding between the ligand alanine carbonyl groups and the 2-amino group of guanine. This is consistent with the observation that the preferred binding site is the sequence CG counter ion effect .
DNA is a negatively charged polyanion attracting counter ions, positively charged Na+, or Ca++ and Mg++ ions as well as basic residues of proteins. The presence of small counter ion affect drug binding, since the counter ions can screen and shield the negative backbone surface allowing non electrolytes as well as positively charged ligand to interact more strongly with the DNA target. High ionic strength, however, reduces non covalent interaction mediated by hydrogen bonds and electrostatic interactions. Role of solvent ligand-receptor binding: There are three general classes of interactions that must be considered in solvated ligandreceptor binding (a) ligand solvent interaction (e.g. hydration shell), (b) Receptor solvent interaction, and (c) ligand-DNA complex with solvent interaction. The three classes basically describe the sequence of events of free ligand interacting with its receptor and the change in overall solvent interaction before and after binding. We have seen that the hydrophobic effect is completely described by this system and the contribution of the entropy of free bulk water is the major driving force of hydrophobic ligand receptor interaction. This type of interaction is found in intercalating substrates because the hydrophobic, aromatic side chains interactive favorably with the aromatic environment of the base pair stacking. The total amount of surface bound water is reduced in the after complex formation. 18.2.2 Rational for drug design: When a compound intercalates into nucleic acids, there are changes which occur in both the DNA and the compound during complex formation that can be used to study the ligand DNA interaction. The binding is of course an equilibrium process because no covalent bond formation is involved. The binding constant can be determined by measuring the free and DNA bound form of the ligand. Since many of the intercalating substrates are aromatic chromophores, this can be done spectroscopically. Also, DNA double helix structures are found to be more stable with intercalating agents present and show a reduced heat denaturation. Correlating these biophysical parameters with cytotoxicity is used to support the antitumor activity of these drugs as based on their ability to intercalate in DNA double helical structures. Improvement of anticancer drugs based on intercalating activity is not only focussed on DNA-ligand interaction, but also on tissue distribution and toxic side effects on the heart (cardiac toxicity) due to redox reduction of the aromatic rings and subsequent free radical formation. Free radical species are thought to induce destructive cellular events such as enzyme inactivation, DNA strand cleavage and membrane lipid peroxidation.
18.2.3 Modeling DNA-ligand interaction of minor groove binders Hairpin minor grove binding molecules have been identified and synthesized that bind to GC reach nucleotide sequences. Hairpin polyamides are linked systems that exploit a set of simple recognition rules for DNA base pairs through specific orientation of imidazole (Im) and pyrrole (Py) rings. The hairpin polyamides originated from the discovery of the three-ring Im-Py-Py molecule that bound to minor groove DNA as an antiparallel side by side dimer.
Fig 74. Structure of hairpin ligand
The compound was found to recognize GC base pairs. Solid phase synthesis of polyamides of variable length has produced efficient ligands, e.g. the eight ring hairpin polyamide ImPyPyPy-g-ImPyPyPy-b-Dp (Dp dimethylamino propylamide) shown in the figure above. This small synthetic molecule has an binding constant in the order of 0.03nM. The optimal goal of polyamide ligand design has been reached with finding structures able to recognize DNA sequences of specific genes. The structure shown above inhibits the expression of 5S RNA in fibroblast cells (skin cancer cells) by interfering with the transcription factor IIIA-binding site. A new strategy of rational drug design exploits the combination of polyamides with bis- intercalating structures. WP631 is a dimeric analog of the clinically proven anthracycline antibiotic daunorobuicin.
Fig75. Structure of WP631.
This new synthetic compound shows an affinity of 10pM and also showed to be resistant against multidrug resistance mechanisms often encountered in antitumor therapy.
Multidrug resistance is a phenomenon where small aromatic compounds are efficiently expelled from the cell by cell membrane transport proteins commonly referred to as ABC transporters (or ATP Binding Cassette proteins). 18.2.4 Drugs that form covalent bonds with DNA targets Drugs that interfere with DNA function by chemically modifying specific nucleotides are Mitomycin C, Cisplatin, and Anthramycin. Mitomycin C is a well characterized antitumor antibiotic which forms a covalent interaction with DNA after reductive activation. The activated antibiotic forms a crosslinking structure between guanine bases on adjacent strands of DNA thereby inhibiting single strand formation (this is essential for mRNA transcription and DNA replication).
Fig 76. Stucture of Mitomycin C
Anthramycin is an antitumor antibiotic which bind covalently to N-2 of guanine located in the minor groove of DNA. Anthramycin has a preference of purine-G-purine sequences (purines are adenine and guanine) with bonding to the middle G.
Fig 77 : a )Anthramycin b) Anthramycin binding to minior groove of DNA
Cisplatin is a transition metal complex cis-diamine-dichloro-platinum and clinically used as anticancer drug. The effect of the drug is due to the ability to platinate the N-7 of guanine on the major groove site of DNA double helix. This chemical modification of platinum atom crosslinks two adjacent guanines on the same DNA strand interfering with the mobility of DNA polymerases.
18.3 METALLOPROTEINS
Metalloprotein is a generic term for a protein that contains a metal cofactor. The metal may be an isolated ion or may be coordinated with a nonprotein organic compound, such
as the porphyrin found in hemoproteins. In some cases, the metal is co-coordinated with a side chain of the protein and an inorganic nonmetallic ion. This kind of protein- metalnonmetal structure is seen in iron-sulfur clusters. Metal ions have a role in a variety of important functions in proteins including protein folding, assembly, stability, conformational change, and catalysis. The presence or absence of a given metal ion is crucial to the conformation or activity of over one third of all proteins. Recent developments have been made in the understanding and design of metal-binding sites in proteins, an important and rapidly advancing area of protein engineering An important class of metalloproteins are metalloenzymes, these are enzymes that contain one or more metal atoms as functional parts of their structures. These metals are often involved in enzyme catalysis, such as in carbonic anhydrase a n d cytochrome c oxidase. Metal ions usually form part of the active site as they can be multicoordinated and thus held in a protein while having a high affinity for the substrate through a lone pair. List of metalloenzymes Ion Cupric Ferrous or Ferric Nickel Selenium Zinc
Examples of enzymes containing this ion Cytochrome oxidase Catalase,Cytochrome(via Heme), Nitrogenase,Hydrogenase Urease Glutathione peroxidase Alcohol dehydrogenase,Carbonic anhydrase DNA polymerase Table 9:Metalloenzymes
18.3.1 Examples Transferrin and lactoferrin, two mammalian iron-binding proteins that control iron levels in mammals. We have determined 3D structures for both proteins, and are now using a combination of crystallography and mutagenesis to understand the factors that control iron binding and release. Hemopexin, a serum protein that binds the heme that is lost during hemolysis (especially in disease) and transports it to the liver for recycling. We have shown that hemopexin binds its heme ligand at the junction of two propeller domains. Embryonic hemoglobins, the earliest hemoglobins produced during human development, which have distinctly different oxygen affinity from the normal adult hemoglobins. Arginase, which plays a central protein in nitrogen metabolism by its regulation of levels of arginine and ornithine, and has a di- manganese centre that is essential for catalysis.
18.3.2 Significance of metal-protein interaction The low dielectric constant characteristic of protein cavities seems to favor the exchange reaction between protein ligand(s) and the metal-bound water. Inner-sphere binding of negatively charged aspartic or glutamic acid residues to metal dications on the protein surface seems unlikely. The magnesium cation has a high affinity for negatively charged acidic residues in sites characterized by a low dielectric constant. However, there is an upper limit in the number of carboxylates that can be exchanged for the metal-bound water molecules: only up to three negatively charged ligands can replace water in the first coordination shell of magnesium. Protonated, acidic residues cannot successfully compete with water for the metal cation. In the isolated carboxylate complexes Mg2+ retains at least one water ligand in its first coordination shell. The lowest-energy ground-state coordination number of Zn2+ bound to 1 acidic or >=2 neutral protein ligands is likely to be 4, in accord with the fact that zinc is 4-coordinate in all structural sites. 5- and 6- (rather than 4-) coordinate structures represent energized states in zinc-containing enzymes x. The observed decrease in the CN of zinc upon protein binding reflects the requirements of the metal and ligands, rather than the constraints of the protein matrix on the metal. It is partly due to the greater charge transfer to Zn2+ from heavy ligand(s) compared to that from water, and the greater charge transfer from a given ligand type to Zn2+ compared to that to Mg2+. During evolution some proteins have chosen Mg2+ as a natural cofactor based mainly on its natural abundance in living cells. Mg-binding sites appear to be weakly protected against other divalent metals like Zn2+, which can replace Mg2+ and, in some cases, inhibit enzymatic activity. Therefore, it seems that it is not the protein that has evolved to select Mg2+ from other cations. Instead, it is the cell machinery that governs the process of metal binding by regulating appropriate concentrations of Mg2+ and other cations (Zn2+ in particular) in various biological compartments. Relative to Mg2+, Zn2+ has a higher affinity for a given protein ligand and strongly prefers a tetrahedral geometry. Consequently, rigid Zn2+-binding sites appear to be more selective than Mg2+-binding sites, and a protein can generally select Zn2+ against the background of a much higher Mg2+ concentration.
18.4 PROTEIN LIGAND INTERACTIONS

18.4.1 Ligand binding sites of immunoglobin: The only category of proteins which has specialized in creating binding sites for practically all kinds of molecules are the immunoglobin .Well studied immunoglobins are the antibodies of the blood plasma and myeloma proteins. The latter are produced by tumor cells. Otherwise they are normal immunoglobins: many myeloma proteins from men and mice have been shown to bind ligand in a reaction which closely resembles that of a specific ligand antibody system. Random pairing of n1 H-chains and n2 L- chains gives rise to n1 . n2 different antigen binding sites.
In both the proteins the ligand binding site is located between the VL domain of the light chain and the VH domain of a heavy chain .This means that the binding site is formed by two different polypeptide chains. The use of two chains might reflect the solution to the problem of forming a practically unlimited number of different binding sites with a limited number of genetic material .If the number of different L- and Hchains is n1 and n2 respectively, then there are n1 n2 combinations of VL and VH domains and therefore n1 n2 different binding sites. The number of different binding sites n1 n2 estimated to be 10 7 accordingly n1 and n2 in the order of 103 to 10 4. There are six hyper variable segments each containing 5 to 10 residues per binding site.The chain fold is very similar in all variable domains of immunoglobin new and immunoglobin McPC 603 and of other immunoglobins (540,543,544) .Structural differences occur predominantly in so-called hyper variable loops, three segments of 5 to 10 each in both VL and VH. The amino acid residues forming the site which is complementary to a given ligand are contributed by these 6 hyper variable segments. The pattern of insertions and deletions characteristic of hyper variable regions determines the dimensions of a binding site. The combining site of I g New consists of a shallow groove of about 15 6 with a depth of about 6 . The phosphoryl choline binding center of I g McPC 603 is approximately 12 deep. 18.4.2 Model Building of immunoglobin Ligand System Structurally known immunoglobins are used to study immunoglobins with other specificities. The VL / VH region of immunoglobins can be regarded as consisting of a rigid framework to which the hyper variable loops are attached. This provides a basis for the comparative analysis of immunoglobin- ligand systems by model building ,using the coordinates of the framework and the amino acid sequences of the hyper variable regions of the immunoglobin which is to be analyzed .The first proteins studied by this approach was the mouse myeloma protein Ig A MOPC 315 which is known to bind 2,4 dinitro phenyl derivatives .A systematic analysis of any immunoglobin ligand system may become possible. The model building studies can be pursued in a systematic manner since the synthesis of antibodies against any specifically designed organic molecule can be induced in animals such as rabbits or goats. Using affinity chromatography the antibodies can then be purified. In general this procedure gives rise to degenerate immune response i.e to the production of several antibodies species by several clones of so called plasma cells. These antibodies differ in affinity constants for the ligand which is also reflected in different chemical spectroscopic and immunologic properties of the binding sites. sequence analysis further revealed that the differences correspond to amino acid changes in the hyper variable regions on the both VL and VH domains. At first glands the degenerate responds seems to complicate the structural elucidation of binding sites. In effect however data on several similar binding sites can be combined and checked against each other. This increases appreciably the probability of finding correct models.
Immunoglobin Ligand systems as models for other Protein Ligand interactions: Our knowledge of protein ligand interactions is still so limited that an X- ray structure is necessary to elucidate them in every given case .This is not a systematic approach since most proteins of interest such as membrane proteins have resisted crystallizing .Therefore it is important to extract the general rules for the ligand binding from the available systems. Immunoglobin ligand complexes are well suited for this purpose because as described above they allow a systematic approach to the problem. Immunoglobin ligand complexes resemble enzyme ligand complexes: There are parallels between the binding properties of immunoglobins and enzymes. As indicated by the values of rate constants (up to 10 8 M-1 sec -1 )the binding step is often a diffusion controlled reaction. The bound ligand has little rotational freedom it is firmly and specifically held by non covalent interactions. The standard free energies of binding range from -6 to -15 kcal/mol which corresponds to dissociation constants of K= 10-4 M to K= 10 -10 M for the ligand enzyme complexes. Ligand binding of Immunoglobin seems to obey the lock and key model. This is also true for many enzymes ligand interactions however, it does not apply to those enzymes which undergo major conformational changes. The Immunoglobin architecture may serve as a basis for in vitro synthesis of peptides with desired binding properties. For theoretical and practical reasons it would be desirable to polypeptide chain in vitro with defined specificity for and affinity to a given compound. One possible approach could start out with a natural or synthetic VL / VH region without hyper variable loops as a framework. By interesting appropriate sequences at the positions of the hyper variable segments a specific binding site for the ligand in question would be modeled without disturbing the folding process and stability of the frame. The example of Cu +2 Zn 2+ Containing super oxide dismutase might be regarded as a natural precedent for this peptide engineering approach. The coordination geometries around the metals at the active site posses very close analogues in crystal structures of copper imidazole complexes. Thus the two basic features of this enzyme are the I g architecture and a metal complex could have been designed by an organic chemist.
18.5 LET US SUM UP

Proteins are selective in their interactions with cell constituents DNA double helix structures are found to be more stable with intercalating agents present and show a reduced heat denaturation. Free radical species are thought to induce destructive cellular events such as enzyme inactivation, DNA strand cleavage and membrane lipid peroxidation Drugs that interfere with DNA function by chemically modifying specific nucleotides are Mitomycin C, Cisplatin, and Anthramycin
An important class of metalloproteins are metalloenzymes, these are enzymes that contain one or more metal atoms as functional parts of their structures

1. Highlight the features of protein lingand interactions. 2. Elaborate on the role of base-fair sequence in DNA Drug interaction.

Give examples of drugs that from covalent bonds wite DNA targets (Refer 18.2.4).

(1) Jonathan Chaires, Michael Waring, (2) Hans-Joachim Bhm (Editor), Gisbert Schneider (Editor), Raimund Mannhold (Series Editor), Hugo Kubinyi (Series Editor), Gerd Folkers (Series Editor) Drug-Nucleic Acid Interactions Protein-Ligand Interactions: From Molecular Recognition to Drug Design
(3) www.iupac.org/publications/pac/1993/pdf/6506x1213.pdf (4) www.scfbio- iitd.res.in/research/target_directed_drugdesign.htm (5) www.scfbio- iitd.res.in/research/target_directed_drugdesign.htm (6) http://metallo.scripps.edu/
LESSON 19 INTERACTION OF PROTEIN WITH LIPIDS AND CARBOHYDRATES

Contents 19.0 Aims and objectives 19.1 Protein-lipid interaction 19.1.1 Introduction 19.1.2 Domains that Bind Proteins and Lipids 19.1.3 Membrane-lipid therapy 19.2 Protein -Carbohydrate Interaction 19.2.1 Introduction 19.2.2 Examples 19.3 Let us sum up 19.4 Lesson End Activities 19.5 Check your Progress 19.6 References

This section will deal with the Protein- lipid interaction and protein-carbohydrate interaction. After reading this unit, you must be able to: Describe mode of interaction between protein and lipid List the domains that bind proteins and lipids Explain membrane lipid therapy Explain interaction between protein and carbohydrate
19.1 PROTEINS -LIPID INTERACTIONS

19.1.1 Introduction Membrane proteins are classified as integral (transmembrane, intrinsic) and peripheral (amphitropic, extrinsic) proteins and both these types of proteins are very sensitive to
changes in their lipid environment. For example, the exchange of sodium ions through the nicotinic acetylcholine receptor (integral protein) is modified by changes in membrane fluidity that can be achieved by altering the membrane lipid composition. On the other hand, PKC (a peripheral protein) translocates from the cytosol to the membrane upon different types of stimuli. One such stimulus is an increase in the non-lamellar phase propensity of membranes (Escrib et al., 1995; Martnez et al., 2005). Thus, a high non- lamellar HII-phase propensity (i.e. elevated negative curvature strain) favors the binding of PKC to membranes and its subsequent activation. Diacylglycerol induces PKC activation not only by binding to the enzyme but also by promoting the HII-phase. In line with this, phorbol esters, which induce a marked activation of PKC enzyme and that are tumor promoters, also induce these non-lamellar phases. For this reason, it has been suggested that inhibitors of PKC could potentially behave as anticancer compounds. However, the anticancer drug minerval also increases the non- lamellar propensity of membrane lipids, activating PKC. An explanation for this apparently paradoxical behavior is that phorbol esters induce massive activation of PKC, followed by rapid enzyme depletion within minutes of the treatment. In contrast, minerval produces a mild (about two- fold) and sustained activation and overexpression of PKC (Martnez et al., 2005). Likewise, some signaling pathways regulated by G proteins are also involved in the control of cell proliferation. In this context, the localization and activity of G proteins is also regulated by the non- lamellar propensity of membranes (Vgler et al., 2004). One important question regarding the regulation of proteinlipid interactions is how lipid structure can control the membrane translocation, cellular localization and the membrane sorting of these and other peripheral signaling proteins. Different mechanisms could be involved in these phenomena based on the formation of membrane defects. Nonlamellar prone lipids (e.g. phosphatidylethanolamine and minerval) reduce phospholipid surface packing (i.e. the lateral pressure) at the interface and the polar regions of the lipid bilayer. This allows hydrophobic domains of peripheral proteins to interact with deep hydrophobic regions of the membrane and/or fatty acid moieties of phospholipids that exit out of the bilayer plane. In addition, electrostatic interactions with phospholipid headgroups and interactions with other proteins also participate in the binding of peripheral proteins to membranes. The effect of lipids in regulating the interaction of these proteins with membranes has been demonstrated using synthetic lipids and purified proteins. Changes in membrane lipid composition have been reported in several pathologies, including cancer. These alterations may be associated with the etiology of the disease or they may reflect an adaptive response. Therefore, lipid interventions could be effective in reversing pathological processes. Despite the potential use of this new therapeutic approach, three important issues will require further study during the following years. First, an in-depth study of the molecular basis underlying the interaction of numerous relevant proteins with membranes will be necessary. Second, the influence of lipid changes on protein activity, pathophysiological processes and therapeutic actions must be defined. Finally, the specificity of clinical strategies based on this principle will have to be assessed. Conventional chemotherapy is based on the design of drugs to target specific proteins and the elucidation of their structure.
From the molecular point of view, conventional chemotherapy and membrane-lipid therapy are quite different, although the final result is the regulation of protein activity. The fact that all membranes contain lipids could question the potential specificity of this therapy. However, there is a huge diversity of membrane lipids, a wide variety of membrane lipid compositions and structures, and tremendous variety among the proteinlipid interactions, which establishes a suitable context to design specific therapies (see above). Obviously, drugs acting through membrane-lipid therapy not only reach pathological cells but also healthy cells, as do drugs acting on a given protein through conventional chemotherapy. However, compounds used in conventional chemotherapy often interact with proteins other than their original target, causing side effects of diverse importance. Thus, the degree of specificity of both approaches could be similar. In this context, the specificity of membrane- lipid therapy is more directly associated with the effects promoted in cancer and other types of pathological cells than with its interaction with a given cell. An example of this is the activity of minerval in tumor cells where its inhibitory effects display an IC50 in the range of 50-100 mM in comparison with the IC50 value of > 5,000 mM in non-tumor human fibroblasts (IMR90 cells). In addition, it has been found that minerval strongly induces the expression or repression of fewer genes than most drugs, further supporting the specificity of membrane- lipid therapy.
19.1.2 Domains that Bind Proteins and Lipids
Fig 78.
PH (pleckstrin homology) domains are about 100 amino acids long and is made up of seven antiparallel b-sheets that form a barrel. The sequence is more variable than SH2 and SH3 domains. pH may mediate protein-protein or proteinlipid interactions
Host Protein B-Adrenergic receptor kinases Phospholipase CdI Akt/PKB
Ligands BG-subunits, PI(4,5)P2 PI(4,5)P2, IP3 PI(3,4)P2, PI(3,4,5)P3
Btk Ras-GAP
PI(3,4,5)P3 PI(3,4,5)P3, I(1,3,4,5)P4
Protein Function Down-regulation of receptors Regulation of enzyme activity A downstream effector of PI 3-kinase, regulation of enzyme activity Regulation of leukocyte activation Link between PI 3-kinase and Ras
Table 8: .Some physiological ligands of PH domains.
19.1.3 Membrane-lipid therapy Definition Clinical drugs that interact with membrane lipids and that modify the composition and structure of cell membranes can change the localization and/or activity of membrane proteins. Several drugs used to combat cancer, cardiovascular pathologies, obesity and other pathologies, regulate membrane lipid structure and they produce a concomitant alteration in the localization and activity of signaling proteins. The net result of these effects is the modulation of certain signaling pathways that reverse the pathological state. Indeed, G proteins, protein kinase C (PKC) and heat shock proteins (HSPs) are among the proteins regulated by membrane-lipid therapy (Escrib et al., 1995; Escrib et al., 1997; Martnez et al., 2005; Torok et al., 2003) and the therapeutic agents used against cancer can inhibit cell proliferation or induce apoptosis and cell differentiation (Martnez
et al., 2005; Martnez et al., 2005b). Regulation of membrane lipid composition and structure is also used to treat cardiovascular pathologies and obesity (Escrib et al., 2003; Alemany et al, 2004; Alemany et al., 2006). Heterogeneity of membrane lipids and lipid structures Membranes are composed of thousands of different lipid molecules that interact dynamically to form the transient or stable structures used by many proteins as platforms for their activity and for their interactions with other proteins. Usually, integral (transmembrane) and peripheral (amphitropic) proteins show important specificities in their interactions with lipids. These preferences may be associated either with a defined type of membrane lipid or a given membrane lipid structure. Most proteins are sensitive to their lipid environment, so that their activity can be modified by changes in membrane lipid composition and structure. These changes in membrane lipid composition and structure may have a physiological basis or they may be a response to external stimuli. An example of the former is the change in cell membrane lipids in response to important changes in water temperature in cold-adapted fishes. Alternatively, one example of the latter is the variation observed in membrane lipids after the intake of a given substance. Membrane lipids can organize into many more secondary structures than proteins and nucleic acids in vitro. Moreover, the number of lipid species exceeds the number of different amino acids and nucleic acid bases by various orders of magnitude. In contrast with proteins and nucleic acids, the spatial relationship between the lipids that form membranes is not defined by covalent bonds, they usually move freely in this environment. Furthermore, since some structural aspects related to membranes still remain unclear, the behavior of lipids is less predictable. However, this does not mean that structure- function relationships are not established in membranes and that bulk thermodynamics can explain all physico-chemical properties of membranes. In fact, changes in the structure of lipids influence not only the physical behavior of membrane lipids (membrane structure) but also the activity of certain associated proteins (membrane function). In general, modifications in membrane lipid structure are reflected in changes in membrane lipid function. Thus, oleic acid (18:1 cisD9) induces a large increase in the non- lamellar (HII) phase propensity of membranes and it alters the interaction and activity of G proteins. In contrast, neither the trans analogue of oleic acid (elaidic acid) nor stearic acids (18:0) markedly influence the lamellar phase of membranes and accordingly, they do not influence G protein activity. During recent years it has become accepted that the fluid mosaic model of membranes described more than 30 years ago by Singer and Nicolson (1972) is a somewhat simplified model which fails to take into account the presence of large membrane domains (e.g. the basal, lateral and apical membrane regions of polarized cells, such as glandular, endothelial and epithelial cells), as wall as the smaller yet highly abundant membrane structure. These domains and structures are characterized by their characteristic lipid and protein composition. In this sense, while the membrane lipid composition most likely defines the presence of specific proteins, proteins may also influence the lipid composition of these domains.
Membrane lipid structure. Lipids with a small polar head, such as phosphatidylethanolamine, have a molecular shape that resembles a truncated cone. They induce a negative curvature strain and favor the organization of membranes into inverted micelles (HII phases) or cubic (bicontinuous) structures. Lipids with a bulky polar head and only one acyl chain (e.g. lysophospholipids, green) have a molecular shape similar to an inverted cone and induce a positive curvature strain in membranes. They favor the formation of tubular (HI) or spheric micelles. Lipids such as phosphatidylcholine have similar cross-sectional areas for the polar head and hydrophobic region and resemble cylinders. They form lamellar phases, with no curvature strain. Lipid bilayers can form a number of different structures, some of them depicted here. Lower panel: In many polarized cells (e.g., small- intestine endothelial cells) there are specialized (e.g. apical, lateral and basal) regions containing specific lipids and proteins. In a given membrane area, GPCR-cluster domains and lipidraft domains can be well differentiated.
Development of anticancer membrane-lipid therapy drugs
It was proposed that the cytotoxic effects of anthracyclines on cancer cells may be exclusively exerted through their interaction with membranes. Indeed, it was later demonstrated that they acted by regulating membrane lipid structure which altered the interaction between peripheral signaling proteins and the plasma membrane. Subsequently, a potential anticancer drug under study, hexamethylene bisacetamide, was found to have a very similar effect on cell membranes, which was associated with the regulation of gene expression. In the knowledge that the mechanism of action was based on the regulation of membrane lipid structure, a number of lipid- interacting molecules were subsequently studied and accordingly, it was demonstrated that oleic acid and its derivatives are potent regulators of the membrane structure. Subsequently, Minerval (the a- hydroxyl-derivative of oleic acid) was found to be a potent antitumor agent without displaying any relevant side effects. Minerval is not the only drug used against cancer that interacts with membranes. As mentioned above, anthracyclines and hexamethylene bisacetamide also regulate membrane structure and peripheral protein-associated signaling. In addition, certain molecules that readily bind to membranes have been shown to have important anticancer effects. For instance, edelfosine [Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac- glycero-3phosphocholine)] and miltefosine [HePC (hexadecyl phosphocholine)] have an important hydrophobic moiety with long hydrocarbon chains (18 and 16 C atoms, respectively), and a polar region comprised of a phosphate group and a choline moiety. This polar-apolar hybrid structure appears to be a common feature of these anticancer drugs that appear to be active in membrane- lipid therapy. This physico-chemical property of these drugs may allow them to interact with both the surface- interface of t h e membrane and with the hydrophobic core, facilitating more stable and long- lasting interactions, as well as inducing the relevant effects on membrane lipid structure. Thus, an interesting class of new anticancer drugs is those compounds known to bind to lipid molecules, such as the molecule NEO6002. This drug results from the combination of gemcitabine with cardiolipin, a phospholipid typical of mitochondrial membranes and it appears to be less toxic and more effective than gemcitabine alone. The lipid modification of gemcitabine induces the membrane- mediated internalization of the compound, which is not blocked by nucleoside transporter inhibitors. Another type of lipid- interacting compound is propofol-DHA, which combines a well-known anesthetic (propofol) with a polyunsaturated fatty acid (docosahexaenoic acid, DHA) that is present in membranes. The resulting compound has been shown to induce apoptosis in MDAMB-231 breast cancer cells. Most cell functions are localized in or around membranes, and lipids control the interaction and activity of many proteins. The relevance of lipids in the treatment of cancer is also highlighted by the lipid abnormalities identified in the membranes of patients with cancer. Thus, changes in the type or abundance of lipids and other types of membrane components may produce either positive or negative effects on health. Hence, membrane-lipid therapy is a new therapeutic approach that could be used in the treatment of cancer and other pathologies.
Membrane-lipid therapy in the treatment of other pathologies Numerous studies indicate that n-3 polyunsaturated and cis- monounsaturated fatty acids have positive effects on cardiovascular health, whereas saturated and transmonounsaturated fatty acids are frequently associated with early onset of a variety of cardiopathies. Indeed, intake of or treatment with monounsaturated fatty acids results in changes in membrane lipid levels with a concomitant regulation in membrane levels of some signaling peripheral proteins, followed by blood pressure reduction in hypertensive subjects and animals. The membrane- lipid therapy approach can also be used to treat other important pathologies, such as obesity, neurodegenerative diseases, infections, etc.
19.2 PROTEIN-CARBOHYDRATE INTERACTIONS

19.2.1 Introduction Many recognition events involve the interactions between an extracellular carbohydrate epitope and a receptor protein. These processes have been observed in such diverse biological processes as fertilization, viral and bacterial infection, inflammatory responses, and the maintenance of lung integrity. Although protein-carbohydrate interactions appear to be important in many biological events, they appear relatively weak when examined experimentally: dissociation constants range from millimolar to micromolar in most cases. Considering the importance of these interactions, it is not understood why the monomeric interactions are so weak. While many different approaches have been used to better understand important factors in protein-carbohydrate interactions, our research efforts have focussed on multivalency and protein engineering. Detailed below are the three subcategories of the protein-carbohydrate interactions project: multivalent ligands in the model system concanavalin A (con A), multivalent inhibitors of Shiga- like toxin 1B (SLT1B), and directed evolution of carbohydrate-binding protein 35 (CBP-35). 19.2.2 Examples Concanavalin A: http://www.chem.duke.edu/~toone/labgroup/5CNAx500.jpgFor the purposes of our ligand-based studies, the protein used is concanavalin A (con A) which is a legume lectin, which is readily obtained from the Jack bean (Canavalia ensiformis). Con A, which can exist as a dimer or tetramer depending on solution pH, binds specifically to mannose. While it is not a medically relevant system, con A- mannoside interactions have been studied and characterized using many different techniques. The binding site of con A has been shown to be a shallow cleft formed by the loop regions of the protein, similar to other legume lectins. There are crystal structures available for con A both free and bound to mannosides, showing the binding sites and minimal epitopes that are bound. In addition to a wealth of structural information, numerous thermodynamic studies have been carried out on this particular lectin. A ligand- modifying strategy for creating glycodendrimers has been utilized to determine the effect of carbohydrate valency on association. The synthesized glycodendrimers have defined valencies and
known structures. Information gathered from the different series (propyl, diglycine, and tetraethylene glycol based) will be pooled with previous observations from other structurally differentiated series to obtain a better understanding of valency effects with regard to binding phenomena.
Fig 79 Structure of Concanavalin A
Shiga-like Toxin 1: http://www.chem.duke.edu/~toone/labgroup/1BOSx500.jpgT h e S h i g a -like toxin 1 (SLT1), a member of the two-component bacterial toxins, adheres to the surfaces of human kidney cells through binding interactions between the protein's pentameric 'B' binding subunit and the glycolipid globotriaosyl ceramide, expressed on cell surfaces. The adherence of the toxin to the cell surface is followed by endocytosis of the toxic warhead 'A' subunit; the 'A' subunit disrupts protein synthesis on the 28 S rRNA subunit by acting as an N-glycosidase. The resulting damage to the kidneys manifests itself as hemorrhagic colitis and in severe cases hemolytic uremic syndrome, for which there is currently no commercially available treatment. Therapeutics based on inhibiting the toxin binding are an attractive yet elusive goal. However, before such therapeutics can be developed a greater understanding of the driving forces of behind high affinity interactions must be gained. Toward that end several members of the lab have been synthesizing multivalent ligands of various types in order to determine important aspects.
Fig 80 Structure of The Shiga-like toxin 1
Carbohydrate-Binding Protein 35: http://www.chem.duke.edu/~toone/labgroup/1A3Kx500.jpgA combination of errorprone PCR and DNA shuffling will be used to generate a library of CBP-35 mutants
which can be displayed on filamentous phage and subsequently selected for affinity or selectivity through the use of a variety of affinity resins.
Fig 81: Carbohydrate-Binding Protein 35
Mutants that show particularly interesting characteristics will be isolated and the protein fully analyzed by calorimetric binding studies to determine dH, KB, and dCp. Thermodynamic solvent isotope effect studies, ELISA and crystallographic experiments may also be carried out in order to better characterize the underlying basis for binding differences. Shown to the left is the x-ray crystallography structure of galectin-3 (the human homologue of CBP-35) complexed with N-acetyllactosamine as determined by J. Rini and co-workers.
19.3 LET US SUM UP

Membrane lipids can organize into many more secondary structures than proteins and nucleic acids in vitro Changes in the type or abundance of lipids and other types of membrane components may produce either positive or negative effects on health. The specificity of membrane- lipid therapy is more directly associated with the effects promoted in cancer and other types of pathological cells than with its interaction with a given cell. The membrane- lipid therapy approach can also be used to treat other important pathologies, such as obesity, neurodegenerative diseases, infections, etc.

Explain the importance of in vitro studies of protein carbohydrate interactions.

Give some examples for protein carbohydrate interaction (Refer 19.2.2)
19.7 REFERENCES
(1) A. Watts (2) JB Swaney Protein-Lipid Interactions Mechanisms of proteinlipid interaction. Association of apolipoproteins A-I and A-II with binary phospholipid mixtures,J. Biol. Chem., Vol. 255, Issue 18, 8791-8797, Sep, 1980 Protein- lipid interactions and the role of water.Horiz Biochem Biophys. 1978;5:241-80. Review. Membrane- lipid therapy: a new approach in molecular medicine,Trends Mol Med. 2006 Jan;12(1):34-43. Epub 2005 Dec 1.
(3) Edelhoch H.
(4) Escrib PV
(5) King D, Lumpkin M, Studying protein-carbohydrate interactions by amide Bergmann C, Orlando R hydrogen/deuterium exchange mass spectrometry Rapid Commun Mass Spectrom. 2002;16(16):1569-74. (6) R. D. Poretz and I. J. Goldstein Protein-carbohydrate interaction XI. A study of turbidity as it relates to concanavalin Aglycan interaction Immunology. 1968 February; 14(2): 165174.
(7) http://www.uib.es/depart/dba/cellbiology/lipid.html
LESSON 20 PIPI INTERACTION AND C-H.PI INTERACTION

Contents 20.0 Aims and objectives 20.1 PiPi interaction 20.2 C-H.Pi interaction 20.3 Let us sum up 20.4 Lesson End Activities 20.5 Check your Progress 20.6 References

In this lesson we have briefly described about the Pi-Pi interactions and C-H Pi interactions. After reading this unit you must be able to: Explain significance of Pi-Pi interactions. Explain significance of C-H Pi interactions.
20.1 PI.PI INTERACTIONS OR AROMATIC INTERACTIONS

In supramolecular chemistry, an aromatic interaction (or - interaction) is a noncovalent interaction between organic compounds containing aromatic moieties. - interactions are caused by intermolecular overlapping of p-orbitals in -conjugated systems, so they become stronger as the number of -electrons increases. - interactions act strongly on flat polycyclic aromatic hydrocarbons such as anthracene, triphenylene, and coronene because of the many delocalized -electrons. This interaction, which is a bit stronger than other noncovalent interactions, plays an important role in various parts of supramolecular chemistry. For example, - interactions have a big influence on molecule-based crystal structures of aromatic compounds. Pi Stacking Interaction or face to face interaction. The rings do not lie directly on top of each other; they are layered in a staggered fashion. Edge-to-face interaction
Aromatic side chain interactions are abundant in proteins. These are predominantly TShaped edge-to- face interactions illustrated to the left. Pi-pi interactions play a key role in the stability and folding of proteins.
20.2 C-H..PI INTERACTIONS

Although the three-dimensional structure of a protein is determined by its covalent structure, i.e. its amino acid sequence, the forces responsible for the folding and stabilization of the structure are mainly non-covalent in nature. These nonconvalent interactions include hydrogen bonds H-bonds) and other electrostatic interactions and the so-called hydrophobic effect. Classical H-bonds, which involve electronegative atoms such as N and O, are well established in biological systems and have been described and reviewed in detail a number of times. Their widespread occurrence manifests itself in the ubiquitous formation of secondarystructure elements such as a-helices and b-sheets and in the fact that nearly all potential H-bond donors and acceptors in the interior of a protein are usually involved in the formation of one or more H-bonds. Energetically, the major terms contributing to the stability of H-bonds are the electrostatic energy and reduced exchange repulsion energy. Polarization and delocalization energy are less important. A set of somewhat weaker interactions has also been recognized to play an important role in protein structure and stability. This set includes N-H and O-H... Pi interactions, interactions between aromatic side-chains and C-H_ O-interactions. Here, the electrostatic contribution to the stability is markedly reduced, but so is the exchange repulsion energy term due to the larger distance between donor and acceptor groups. The overall stabilization energy is therefore much smaller, but still significant. The even weaker C-H...Pi- interactions have also been described to occur. These interactions can be characterized by a close to negligible contribution of the electrostatic energy term to the overall stabilization energy, whereas the delocalization energy term far outweighs all other energy terms. As a consequence, C-H...Pi- interactions are stable in both polar and non-polar solvents. The overall stabilization energy of about 0.5-1.0 kcal/mol per interaction is enough to make it a potentially important contributor to the overall protein stability, which in many cases is not more than a few kcal/mol itself. The cases in which C-HPi- interactions have been described in proteins include the formation of complexes of proteins with special ligands or cofactors such as the heme group, pyridoxal-50-phosphate, nucleotides, carbohydrates and bound peptides, or special geometric circumstances, for instance between neighboring side-chains around a cis peptide bond. The importance of this interaction has also been recognized in the design of serine protease inhibitors, but no study exists to date that deals with the occurrence of C-HPi- interactions within the protein context itself. In contrast to the situation involving biological systems, C-HPi- interactions have been well established in organic chemistry since 1952 when Tamres observed that benzene and analogous compounds dissolve exothermically in chloroform. Five years later, Reeves and Schneider showed by NMR that this interaction was a type of H-bond.
Since then, C-HPi- interactions have been described in vast number of small molecule systems from simple organic and aromatic compounds and inclusion complexes. An excellent treatise of these observations can be found in the recently published book by Nishio et al. (1998). Here, we used a non-redundant data base of 1154 protein structures to examine systematically and for the first time, the occurrence and the role C-HPiinteractions in protein structures. We will demonstrate that these interactions can be classified as weak H-bonds, and that they are likely to play an important role in protein stability. The location of the respective interactions within the protein structures was assessed by calculating their radial distribution in a sphere. The center of the sphere is defined by the center-of- mass of the protein and the radius by the distance of the center- of- mass to the atom which is farthest away it. This whole sphere was divided in volume shells of equal thickness and the number of interactions in each shell was counted. As can be expected from their hydrophobicity, interactions involving aromatic groups are found in general closer to the center of the protein than interactions involving the more polar p-systems. Counting all interactions with aromatic acceptor groups, the largest number interactions is found in shell 5 (the total number shells is ten) from the center, whereas for the other acceptor groups the largest number is observed in shell 6. One has to bear in mind, however, that this method works well only when the structure is strictly spherical. Furthermore, due to the large number of interactions, the method is not very sensitive, but the overall trend which could be discerned makes sense. The fraction solvent accessibility for each interaction pair was also calculated. This method yields basically the same result. The average solvent accessibility for all interaction pairs with aromatic acceptors is 15%, whereas it is 27 %, 30 %, and 30% for the interaction pairs involving amide, carboxylate, and guanidinium groups as pacceptors, respectively. One has to be careful in evaluating these data in terms of the relative location, since only complete amino acid residues are taken into account. An arginine residue for instance can have fraction of 20% solvent-accessible surface and still be on the surface with just the guanidinium group exposed to solvent. For this reason any statement on the overall location is of limited use, and as above, only general trends can be observed. However, these overall trends are important. C-HPi- interactions have been identified to occur ubiquitously in almost all proteins. The most prominent representatives are the interactions between aromatic C-H donor groups and aromatic p-acceptors and the interactions between aliphatic C-H donor groups and aromatic p-acceptors, although the other ten classes occur quite frequently as well. The geometric parameters calculated for these interactions suggest that C-HPiinteractions can be classified as weak H-bonds, rather than relatively unspecific hydrophobic interactions. C-HPi- interactions involving aromatic p-systems as acceptor groups are generally found closer to the center of the protein than the interactions with the more polar p-systems, the amide, the acid, and the guanidium groups. No such distinction can be made on the basis of the different C-H-donor groups.
C-HPi- interactions appear to occur relatively frequently between side-chains of residues that are not very far apart in sequence. In some cases they might even be responsible for the stabilization of structural elements such as 3.10- helices or nonproline cis peptide bonds. The conservation of amino acid residues with p-systems may in some cases be linked to their involvement in C-HPi- interactions and to the stability or the function.
20.3 LET US SUM UP

- interactions are caused by intermolecular overlapping of p-orbitals in conjugated systems, so they become stronger as the number of -electrons increases. - interactions act strongly on flat polycyclic aromatic hydrocarbons such as anthracene, triphenylene, and coronene because of the many delocalized electrons. Pi-pi interactions play a key role in the stability and folding of proteins C-HPi- interactions involving aromatic p-systems as acceptor groups are generally found closer to the center of the protein than the interactions with the more polar p-systems, the amide, the acid, and the guanidium groups

How do you prove that pi-pi interaction are more significant in molecular chemistry?

Give some advantages of Pi-Pi interaction (Refer 20.1).
20.6 REFERENCES
(1) Tsuzuki S, Honda K, Uchimaru T, Mikami M, Tanabe K. Origin of attraction and directionality of the pi/pi interaction: model chemistry calculations of benzene dimer interaction, J Am Chem Soc. 2002 Jan 9;124(1):104-12 Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant.Biochemistry. 2003 Jun 10;42(22):6853-62. C-H...pi- interactions in proteins, J Mol Biol. 2001 Mar Jabs 16;307(1):357-77
(2) Yanagisawa S et.al
(3) Brandl M, Weiss MS, A, Shnel J, lgenfeld R.
(4) http://www.tim.hi- ho.ne.jp/dionisio/ http://www.chemsoc.org/ExemplarChem/entries/2004/warwick_robinson/Pi.htm
LESSON 21- ULTRAVIOLET-VISIBLE SPECTROSCOPY

Contents 21.0 Aims and objectives 21.1 Introduction 21.2 Beer-Lambert law 21.3 Ultraviolet-visible spectrophotometer 21.4 Ultraviolet-visible spectrum 21.5 Applications 21.6 Let us sum up 21.7 Lesson End Activities 21.8 Check your Progress 21.9 References

In this lesson we have described about the Ultraviolet visible spectroscopy and its applications After reading this lesson you will be able to: Understand the basic principles of infra-red absorption and spectroscopy Understand Beer-Lambert law List the applications of Ultraviolet visible spectroscopy
21.1 INTRODUCTION
The Light of Knowledge is an often used phrase, but it is particularly appropriate in reference to spectroscopy. Most of what we know about the structure of atoms and molecules comes from studying their interaction with light (electromagnetic radiation). Different regions of the electromagnetic spectrum provide different kinds of information as a result of such interactions. Realizing that light may be considered to have both wavelike and particle- like characteristics, it is useful to consider that a given frequency or wavelength of light is associated with a "light quanta" of energy we now call a photon. As noted in the following equations, frequency and energy change proportionally, but wavelength has an inverse relationship to these quantities.
In order to "see" a molecule, we must use light having a wavelength smaller than the molecule itself (roughly 1 to 15 angstrom units). Such radiation is found in the X-ray
region of the spectrum, and the field of X-ray crystallography yields remarkably detailed pictures of molecular structures amenable to examination. The chief limiting factor here is the need for high quality crystals of the compound being studied. The methods of Xray crystallography are too complex to be described here; nevertheless, as automatic instrumentation and data handling techniques improve, it will undoubtedly prove to be the procedure of choice for structure determination. The spectroscopic techniques described below do not provide a three-dimensional picture of a molecule, but instead yield information about certain characteristic features. A brief summary of this information follows: Mass Spectrometry: Sample molecules are ionized by high energy electrons. The mass to charge ratio of these ions is measured very accurately by electrostatic acceleration and magnetic field perturbation, providing a precise molecular weight. Ion fragmentation patterns may be related to the structure of the molecular ion. Ultraviolet-Visible Spectroscopy: Absorption of this relatively high-energy light causes electronic excitation. The easily accessible part of this region (wavelengths of 200 to 800 nm) shows absorption only if conjugated pi-electron systems are present. Infrared Spectroscopy: Absorption of this lower energy radiation causes vibrational and rotational excitation of groups of atoms within the molecule. Because of their characteristic absorptions identification of functional groups is easily accomplished. Nuclear Magnetic Resonance Spectroscopy: Absorption in the low-energy radio- frequency part of the spectrum causes excitation of nuclear spin states. NMR spectrometers are tuned to certain nuclei (e.g. 1 H, 13 C, 19F & 31 P). For a given type of nucleus, high-resolution spectroscopy distinguishes and counts atoms in different locations in the molecule. Ultraviolet- visible spectroscopy or ultraviolet-visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons and spectrophotometry. It uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy space molecules undergo electronic transitions.
Fig 82 . Electromagnetic Spectrum
21.2 BEER-LAMBERT LAW

The method is most often used in a quantitative way to determine concentrations of an absorbing species in solution, using the Beer-Lambert law:
where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the concentration of the absorbing species. For each species and wavelength, is a constant known as the molar absorptivity or extinction coefficient. This constant is a fundamental molecular property in a given solvent, at a particular temperature and pressure, and has units of 1 / M * cm or often AU / M * cm.The absorbance and extinction are sometimes defined in terms of the natural logarithm instead of the base-10 logarithm. The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances. A second order polynomial relationship between absorption and concentration is sometimes encountered for very large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red, for example).
21.3 ULTRAVIOLET-VISIBLE SPECTROPHOTOMETER

The instrument used in ultraviolet-visible spectroscopy is called a UV/vis spectrophotometer. It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance:
A = - log(%T) The basic parts of a spectrophotometer are a light source (often an incandescent bulb for the visible wavelengths, or a deuterium arc lamp in the ultraviolet), a holder for the sample, a diffraction grating or monochromator to separate the different wavelengths of light, and a detector. The detector is typically a photodiode or a CCD. Photodiodes are used with monochromators, which filter the light so that only light of a single wavelength reaches the detector. Diffraction gratings are used with CCDs, which collects light of different wavelengths on different pixels.
Fig 83 . S pectrophotometer
A spectrophotometer can be either single beam or double beam. In a single beam instrument (such as the Spectronic 20), all of the light passes through the sample cell. Io must be measured by removing the sample. This was the earliest design, but is still in common use in both teaching and industrial labs. In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. Some double-beam instruments have two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The detector alternates between measuring the sample beam and the reference beam. Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of gases and even of solids can also be measured. Samples are typically placed in a
transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm. (This width becomes the path length, L, in the Beer-Lambert law.) Test tubes can also be used as cuvettes in some instruments. The best cuvettes are made of high quality quartz, although glass or plastic cuvettes are common. (Glass and most plastics absorb in the UV, which limits their usefulness to visible wavelengths.)
21.4 ULTRAVIOLET-VISIBLE SPECTRUM

An ultraviolet-visible spectrum is essentially a graph of light absorbance versus wavelength in a range of ultraviolet or visible regions. Such a spectrum can often be produced directly by a more sophisticated spectrophotometer, or the data can be collected one wavelength at a time by simpler instruments. Wavelength is often represented by the symbol . Similarly, for a given substance, a standard graph of the extinction coefficient () vs. wavelength () may be made or used if one is already available. Such a standard graph would be effectively "concentration-corrected" and thus independent of concentration. For the given substance, the wavelength at which maximum absorption in the spectrum occurs is called max, pronounced "Lambda- max". The Woodward-Fieser rules are a set of empirical observations which can be used to predict max, the wavelength of the most intense UV/Vis absorption, for conjugated organic compounds such as dienes and ketones. This spectrum can be used qualitatively to identify components in a sample as each component has their own unique absorbance spectrum (like a fingerprint).
21.5 APPLICATIONS
a)UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds. b)Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. The color of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands. For instance, the color of a dilute solution of copper sulphate is a very light blue; adding ammonia intensifies the color and changes the wavelength of maximum absorption (_max). c)Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol for organicsoluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.)
d)While charge transfer complexes also give rise to colors, the colors are often too intense to be used for quantitative measurement. e)The Beer-Lambert law states that the absorbance of a solution is directly proportional to the solution's concentration. Thus UV/VIS spectroscopy can be used to determine the concentration of a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve. f) UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response which can be assumed to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; this is very similar to the use of calibration curves. The response (e.g., peak height) for a particular concentration is known as the response factor.
21.6 LET US SUM UP

Ultraviolet- visible spectroscopy or ultraviolet-visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons and spectrophotometry. The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a universal relationship for the concentration and absorption of all substances.

Write down the applications and features of ultraviolet spectroscopy.

Make a study and analyze the significance of Bear-Lamberts Law. (Refer 21.2).
21.9 REFERENCES
(1) http://en.wikipedia.org/wiki/UV/VIS_spectroscopy (2) http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm (3) http://www.chemsoc.org/pdf/LearnNet/rsc/UV_txt.pdf
LESSON 22- INFRARED SPECTROSCOPY

Contents 22.0 Aims and objectives 22.1 Introduction 22.2 Theory 22.3 Sample preparation 22.4 Typical method 22.5 Isotope effects 22.6 Fourier transform infrared spectroscopy 22.7 Two-dimensional infrared spectroscopy 22.8 Uses and applications 22.9 Let us sum up 22.10 Lesson End Activities 22.11 Check your Progress 22.12 References

In this lesson we have described about infrared visible spectroscopy and its applications After reading this lesson you must be able to: Describe the theory for infrared visible spectroscopy Know the method of sample preparation Know different types of infrared visible spectroscopy Know the applications of infrared visible spectroscopy
22.1 INTRODUCTION
Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify compounds or investigate sample composition
22.2 THEORY
Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they rotate or vibrate corresponding to discrete energy levels. These resonant frequencies are determined by the shape of the molecular potential energy surfaces, the masses of the atoms and, by the associated vibronic coupling. In order for a vibrational mode in a molecule to be IR active, it must be associated with changes in the permanent dipole. In particular, in the Born-Oppenheimer and harmonic approximations, i.e. when the molecular Hamiltonian corresponding to the electronic ground state can be approximated by a harmonic oscillator in the neighborhood of the equilibrium molecular geometry, the resonant frequencies are determined by the normal modes corresponding to the molecular electronic ground state potential energy surface. Nevertheless, the resonant frequencies can be in a first approach related to the strength of the bond, and the mass of the atoms at either end of it. Thus, the frequency of the vibrations can be associated with a particular bond type. Simple diatomic molecules have only one bond, which may stretch. More complex molecules have many bonds, and vibrations can be conjugated, leading to infrared absorptions at characteristic frequencies that may be related to chemical groups. For example, the atoms in a CH2 group, commonly found in organic compounds can vibrate in six different ways, symmetrical and antisymmetrical stretching, scissoring, rocking, wagging and twisting; as shown below
Fig 84. Types of Vibration
The infrared spectrum of a sample is collected by passing a beam of infrared light through the sample. Examination of the transmitted light reveals how much energy was absorbed at each wavelength. This can be done with a monochromatic beam, which changes in wavelength over time, or by using a Fourier transform instrument to measure
all wavelengths at once. From this, a transmittance or absorbance spectrum can be produced, showing at which IR wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details about the molecular structure of the sample. This technique works almost exclusively on samples with covalent bonds. Simple spectra are obtained from samples with few IR active bonds and high levels of purity. More complex molecular structures lead to more absorption bands and more complex spectra. The technique has been used for the characterization of very complex mixtures however.
22.3 SAMPLE PREPARATION

Gaseous samples require little preparation beyond purification, but a sample cell with a long pathlength (typically 5-10 cm) is normally needed, as gases show relatively weak absorbances. Liquid samples can be sandwiched between two plates of a high purity salt (commonly sodium chloride, or common salt, although a number of other salts such as potassium bromide or calcium fluoride are also used). The plates are transparent to the infrared light and will not introduce any lines onto the spectra. Some salt plates are highly soluble in water, and so the sample, washing reagents and the like must be anhydrous (without water). Solid samples can be prepared in two major ways. The first is to crush the sample with a mulling agent (usually nujol) in a marble or agate mortar, with a pestle. A thin film of the mull is applied onto salt plates and measured. The second method is to grind a quantity of the sample with a specially purified salt (usually potassium bromide) finely (to remove scattering effects from large crystals). This powder mixture is then crushed in a mechanical die press to form a translucent pellet through which the beam of the spectrometer can pass. It is important to note that spectra obtained from different sample preparation methods will look slightly different from each other due to the different physical states the sample is in. 2.4 Typical method
Fig 85.Apparatus
A beam of infrared light is produced and split into two separate beams. One is passed through the sample, the other passed through a reference which is often the substance the sample is dissolved in. The beams are both reflected back towards a detector, however first they pass through a splitter which quickly alternates which of the two beams enters the detector. The two signals are then compared and a printout is obtained. A reference is used for two reasons: This prevents fluctuations in the output of the source affecting the data This allows the effects of the solvent to be cancelled out (the reference is usually a pure form of the solvent the sample is in)
Fig 86. Summary of absorptions of bonds in organic molecules (Wavenumbers listed in cm-1).
22.5 ISOTOPE EFFECTS

The different isotopes in a particular species may give fine detail in infrared spectroscopy. For example, the O-O stretching frequency of oxyhemocyanin is experimentally determined to be 832 and 788 cm-1 for (16O-16O) and (18O-18O) respectively. By considering the O-O as a spring, the wavelength of absorbance, can be calculated:
Where k is the spring constant for the bond, and is the reduced mass of the A-B system:
(mi is the mass of atom i). The reduced masses for 16O-16O and 18O-18O can be approximated as 8 and 9 respectively. Thus
22.6 FOURIER TRANSFORM INFRARED SPECTROSCOPY

Fourier transform infrared (FTIR) spectroscopy is a measurement technique for collecting infrared spectra. Instead of recording the amount of energy absorbed when the frequency of the infra-red light is varied (monochromator), the IR light is guided through an interferometer. After passing the sample the measured signal is the interferogram. Performing a mathematical Fourier transform on this signal results in a spectrum identical to that from conventional (dispersive) infrared spectroscopy. FTIR spectrometers are cheaper than conventional spectrometers because building of interferometers is easier than the fabrication of a monochromator. In addition, measurement of a single spectrum is faster for the FTIR technique because the information at all frequencies is collected simultaneously. This allows multiple samples to be collected and averaged together resulting in an improvement in sensitivity. Because of its various advantages, virtually all modern infrared spectrometers are FTIR instruments.
22.7 TWO-DIMENSIONAL INFRARED SPECTROSCOPY

Two-dimensional infrared correlation spectroscopy analysis is the application of 2D correlation analysis on infrared spectra. By extending the spectral information of a perturbed sample, spectral analysis is simplified and resolution is enhanced. The 2D synchronous and 2D asynchronous spectra represent a graphical overview of the spectral changes due to a perturbation (such as a changing concentration or changing temperature) as well as the relationship between the spectral changes at two different wavenumbers.
Nonlinear two-dimensional infrared spectroscopy is the infrared version of correlation spectroscopy. Nonlinear two-dimensional infrared spectroscopy is a technique that has become available with the development of femtosecond infrared laser pulses. In this experiment first a set of pump pulses are applied to the sample. This is followed by a waiting time, where the system is allowed to relax. The waiting time typically lasts from zero to several picoseconds and the duration can be controlled with a resolution of tens of femtoseconds. A probe pulse is then applied resulting in the emission of a signal from the sample. The nonlinear two-dimensional infrared spectrum is a two-dimensional correlation plot of the frequency 1 that was excited by the initial pump pulses and the frequency 3 excited by the probe pulse after the waiting time. This allows the observation of coupling between different vibrational modes. Because of its extremely high time resolution it can be used to follow changes in molecular configurations taking place on a picosecond timescale. It is still a largely unexplored technique and is becoming increasingly popular for fundamental research. Like in two-dimensional nuclear magnetic resonance (2DNMR) spectroscopy this technique spreads the spectrum in two dimensions and allow for the observation of cross peaks that contain information on the coupling between different modes. In contrast to 2DNMR nonlinear two-dimensional infrared spectroscopy also involve the excitation to overtones. These excitations result in excited state absorption peaks located below the diagonal and cross peaks. In 2DNMR two distinct techniques, COSY and NOESY, are frequently used. The cross peaks in the first are related to the scalar coupling, while in the later they are related to the spin transfer between different nuclei. In nonlinear twodimensional infrared spectroscopy analogs have been drawn to these 2DNMR techniques. Nonlinear two-dimensional infrared spectroscopy with zero waiting time corresponds to COSY and nonlinear two-dimensional infrared spectroscopy with finite waiting time allowing vibrational population transfer corresponds to NOESY. The COSY variant of nonlinear two-dimensional infrared spectroscopy has been used for determination of the secondary structure content proteins.
22.8 USES AND APPLICATIONS

Infrared spectroscopy is widely used in both research and industry as a simple and reliable technique for measurement, quality control and dynamic measurement. The instruments are now small, and can be transported, even for use in field trials. With increasing technology in computer filtering and manipulation of the results, samples in solution can now be measured accurately (water produces a broad absorbance across the range of interest, and thus renders the spectra unreadable without this computer treatment). Some machines will also automatically tell you what substance is being measured from a store of thousands of reference spectra held in storage. By measuring at a specific frequency over time, changes in the character or quantity of a particular bond can be measured. This is especially useful in measuring the degree of polymerization in polymer manufacture. Modern research machines can take infrared measurements across the whole range of interest as frequently as 32 times a second. This
can be done whilst simultaneous measurements are made using other techniques. This makes the observations of chemical reactions and processes quicker and more accurate. Techniques have been developed to assess the quality of tea- leaves using infrared spectroscopy. This will mean that highly trained experts (also called 'noses') can be used more sparingly, at a significant cost saving. Infrared spectroscopy has been highly successful for applications in both organic and inorganic chemistry. Infrared spectroscopy has also been successfully utilized in the field of semiconductor microelectronics: for example, infrared spectroscopy can be applied to semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous silicon, silicon nitride, etc.
22.9 LET US SUM UP

Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they rotate or vibrate corresponding to discrete energy level Two-dimensional infrared correlation spectroscopy analysis is the application of 2D correlation analysis on infrared spectra.

If spectroscopy in widely used them UV spectroscopy Give your views on this statement.

Explain and know the importance of isotope effects on IR spectroscopy.

(1) http://en.wikipedia.org/wiki/Infrared_spectroscopy (2) http://www.chemsoc.org/pdf/LearnNet/rsc/UV_txt.pdf (3) http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/InfraRed/infrared.htm
LESSON 23 - NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Contents 23.0 Aims and objectives 23.1 Introduction 23.2 Nuclear spin and the splitting of energy levels in a magnetic field 23.3 Calculating transition energy 23.4 The absorption of radiation by a nucleus in a magnetic field 23.5 Relaxation processes 23.5.1 Spin - lattice relaxation 23.5.2 Spin - spin relaxation 23.6 Basic NMR Techniques 23.6.1 Chemical Shift 23.6.2 J-coupling 23.7 Correlation spectroscopy 23.8 Solid-State nuclear magnetic resonance 23.9 Applications 23.9.1 NMR spectroscopy applied to proteins 23.9.2 Medicine 23.9.3 Chemistry 23.9.4 Non-destructive testing 23.9.5 Data acquisition in the petroleum industry 23.9.6 Process control 23.9.7 Earth's field NMR 23.9.8 Magnetometers 23.10 Let us sum up 23.11 Lesson End Activities 23.12 Check your Progress 23.13 References
23.0 AIM AND OBJECTIVES

This section will deal with the basic NMR techniques and its applications. After reading this lesson, you will be able to: Know the concept behind NMR spectroscopy. Know how to calculate transition energy. Explain the relaxation process. List the applications of NMR spectroscopy..
23.1 INTRODUCTION
Nuclear Magnetic Resonance spectroscopy is a powerful and theoretically complex analytical tool. On this page, we will cover the basic theory behind the technique. It is important to remember that, with NMR, we are performing experiments on the nuclei of atoms, not the electrons. The chemical environment of specific nuclei is deduced from information obtained about the nuclei.
23.2 NUCLEAR SPIN AND THE SPLITTING OF ENERGY LEVELS IN A MAGNETIC FIELD
Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes. In many atoms (such as 12 C) these spins are paired against each other, such that the nucleus of the atom has no overall spin. However, in some atoms (such as 1 H and 13 C) the nucleus does possess an overall spin. The rules for determining the net spin of a nucleus are as follows; 1. If the number of neutrons and the number of protons are both even, then the nucleus has NO spin. 2. If the number of neutrons plus the number of protons is odd, then the nucleus has a half- integer spin (i.e. 1/2, 3/2, 5/2) 3. If the number of neutrons and the number of protons are both odd, then the nucleus has an integer spin (i.e. 1, 2, 3) The overall spin, I, is important. Quantum mechanics tells us that a nucleus of spin I will have 2I + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations. In the absence of an external magnetic field, these orientations are of equal energy. If a magnetic field is applied, then the energy levels split. Each level is given a magnetic quantum number, m.
Fig 87: Energy levels for a nucleus
When the nucleus is in a magnetic field, the initial populations of the energy levels are determined by thermodynamics, as described by the Boltzmann distribution. This is very important, and it means that the lower energy level will contain slightly more nuclei than the higher level. It is possible to excite these nuclei into the higher level with electromagnetic radiation. The frequency of radiation needed is determined by the difference in energy between the energy levels.
23.3 CALCULATING TRANSITION ENERGY

The nucleus has a positive charge and is spinning. This generates a small magnetic field. The nucleus therefore possesses a magnetic moment, , which is proportional to its spin,I.
The constant, , is called the magnetogyric ratio and is a fundamental nuclear constant which has a different value for every nucleus. h is Plancks constant. The energy of a particular energy level is given by;
Where B is the strength of the magnetic field at the nucleus. The difference in energy between levels (the transition energy) can be found from
This means that if the magnetic field, B, is increased, so is E. It also means that if a nucleus has a relatively large magnetogyric ratio, then E is correspondingly large.
If you had trouble understanding this section, try reading the next bit (The absorption of radiation by a nucleus in a magnetic field) and then come back.
23.4 THE ABSORPTION OF RADIATION BY A NUCLEUS IN A MAGNETIC FIELD

In this discussion, we will be taking a "classical" view of the behaviour of the nucleus that is, the behaviour of a charged particle in a magnetic field. Imagine a nucleus (of spin 1/2) in a magnetic field. This nucleus is in the lower energy level (i.e. its magnetic moment does not oppose the applied field). The nucleus is spinning on its axis. In the presence of a magnetic field, this axis of rotation will precess around the magnetic field;
Fig:88 Nucleus (of spin 1/2) in a magnetic field
The frequency of precession is termed the Larmor frequency, which is identical to the transition frequency. The potential energy of the precessing nucleus is given by; E = - B cos where is the angle between the direction of the applied field and the axis of nuclear rotation. If energy is absorbed by the nucleus, then the angle of precession, , will change. For a nucleus of spin 1/2, absorption of radiation "flips" the magnetic moment so that it opposes the applied field (the higher energy state).
Fig:89.
It is important to realise that only a small proportion of "target" nuclei are in the lower energy state (and can absorb radiation). There is the possibility that by exciting these nuclei, the populations of the higher and lower energy levels will become equal. If this occurs, then there will be no further absorption of radiation. The spin system is saturated. The possibility of saturation means that we must be aware of the relaxation processes which return nuclei to the lower energy state.
23.5 RELAXATION PROCESSES

How do nuclei in the higher energy state return to the lower state? Emission of radiation is insignificant because the probability of re-emission of photons varies with the cube of the frequency. At radio frequencies, re-emission is negligible. We must focus on nonradiative relaxation processes (thermodynamics!). Ideally, the NMR spectroscopist would like relaxation rates to be fast - but not too fast. If the relaxation rate is fast, then saturation is reduced. If the relaxation rate is too fast, linebroadening in the resultant NMR spectrum is observed. There are two major relaxation processes;

Spin - lattice (longitudinal) relaxation Spin - spin (transverse) relaxation
23.5.1 Spin - lattice relaxation Nuclei in an NMR experiment are in a sample. The sample in which the nuclei are held is called the lattice. Nuclei in the lattice are in vibrational and rotational motion, which creates a complex magnetic field. The magnetic field caused by motion of nuclei within the lattice is called the lattice field. This lattice field has many components. Some of these components will be equal in frequency and phase to the Larmor frequency of the nuclei of interest. These components of the lattice field can interact with nuclei in the higher energy state, and cause them to lose energy (returning to the lower state). The
energy that a nucleus loses increases the amount of vibration and rotation within the lattice (resulting in a tiny rise in the temperature of the sample). The relaxation time, T1 (the average lifetime of nuclei in the higher energy state) is dependant on the magnetogyric ratio of the nucleus and the mobility of the lattice. As mobility increases, the vibrational and rotational frequencies increase, making it more likely for a component of the lattice field to be able to interact with excited nuclei. However, at extremely high mobilities, the probability of a component of the lattice field being able to interact with excited nuclei decreases. 23.5.2 Spin - spin relaxation Spin - spin relaxation describes the interaction between neighbouring nuclei with identical precessional frequencies but differing magnetic quantum states. In this situation, the nuclei can exchange quantum states; a nucleus in the lower energy level will be excited, while the excited nucleus relaxes to the lower energy state. There is no net change in the populations of the energy states, but the average lifetime of a nucleus in the excited state will decrease. This can result in line-broadening.
Fig 90: B asic arrangement of an NMR spectrometer
The basic arrangement of an NMR spectrometer is shown in the diagram above. The sample is positioned in the magnetic field and excited via pulsations in the radio frequency input circuit. The realigned magnetic fields induce a radio signal in the output circuit which is used to generate the output signal. Fourier analysis of the complex output produces the actual spectrum. The pulse is repeated as many times as necessary to allow the signals to be identified from the background noise.
23.6 BASIC NMR TECHNIQUES

When placed in a magnetic field, NMR active nuclei (such as 1H or 13C) absorb at a frequency characteristic of the isotope. The resonant frequency, energy of the absorption
and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 tesla magnetic field, protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength. In the Earth's magnetic field the same nuclei resonate at audio frequencies. This effect is used in Earth's field NMR spectrometers and other instruments. Because these instruments are portable and inexpensive, they are often used for teaching and field work 23.6.1 Chemical Shift Depending on the local chemical environment, different protons in a molecule resonate at slightly different frequencies. Since this frequency shift is proportional to the strength of the magnetic field, it is converted into a field- independent dimensionless value known as the chemical shift. The chemical shift is reported as a relative measure from some reference resonance frequency.For the nuclei1H,13C, and 29Si,TMS (tetramethylsilane) is commonly used as a reference.This difference between the frequency of the signal and the frequency of the reference is divided by frequency of the reference signal to give the chemical shift. The frequency shifts are extremely small in comparison to the fundamental NMR frequency. A typical frequency shift might be 100 Hz, compared to a fundamental NMR frequency of 100 MHz, so the chemical shift is generally expressed in parts per million (ppm). By understanding different chemical environments, the chemical shift can be used to obtain some structural information about the molecule in a sample. The conversion of the raw data to this information is called assigning the spectrum. For example, for 1H-NMR spectrum for ethanol (CH3 CH2 OH) one would expect three specific signals at three specific chemical shifts. One for the CH3 group, one for the CH2 group and one for the OH. A typical CH3 group has a shift around 1 ppm, the CH2 attached to a OH has a shift of around 4 ppm and the OH has a shift around 23 ppm depending on the solvent used. Because of molecular motion at room temperature, the three methyl protons average out during the course of the NMR experiment (which typically requires a few ms). These protons become degenerate and form a peak at the same chemical shift. The shape and size of peaks are indicators of chemical structure too. In the example abovethe proton spectrum of ethanolthe CH3 peak would be three times as large as the OH. Similarly the CH2 peak would be twice the size of the OH peak but only 2/3 the size of the CH3 peak. Modern analysis software allows analysis of the size of peaks to understand how many protons give rise to the peak. This is known as integrationa mathematical process which calculates the area under a graph (essentially what a spectrum is). The analyst must integrate the peak and not measure its height because the peaks also have widthand thus its size is dependent on its area not its height. However, it should be mentioned that the number of protons, or any other observed nucleus, is only proportional to the
intensity, or the integral, of the NMR signal, in the very simplest one-dimensional NMR experiments. In more elaborate experiments, for instance, experiments typically used to obtain carbon-13 NMR spectra, the integral of the signals depends on the relaxation rate of the nucleus, and its scalar and dipolar coupling constants. Very often these factors are poorly understood - therefore, the integral of the NMR signal is very difficult to interpret in more complicated NMR experiments. 23.6.2 J-coupling Some of the most useful information for structure determination in a one-dimensional NMR spectrum comes from J-coupling or scalar coupling (a special case of spin-spin coupling) between NMR active nuclei. This coupling arises from the interaction of different spin states through the chemical bonds of a molecule and results in the splitting of NMR signals. These splitting patterns can be complex or simple and, likewise, can be straightforwardly interpretable or deceptive. This coupling provides detailed insight into the connectivity of atoms in a molecule. Coupling to n equivalent (spin ) nuclei splits the signal into a n+1 multiplet with intensity ratios following Pascal's triangle as described on the right. Coupling to additional spins will lead to further splittings of each component of the multiplet e.g. coupling to two different spin nuclei with significantly different coupling constants will lead to a doublet of doublets (abbreviation: dd). Note that coupling between nuclei that are chemically equivalent (that is, have the same chemical shift) has no effect of the NMR spectra and couplings between nuclei that are distant (usually more than 3 bonds apart for protons in flexible molecules) are usually too small to cause observable splittings. Long-range couplings over more than three bonds can often be observed in cyclic and aromatic compounds, leading to more complex splitting patterns. For example, in the proton spectrum for ethanol described above, the CH3 group is split into a triplet with an intensity ratio of 1:2:1 by the two neighboring CH2 protons. Similarly, the CH2 is split into a quartet with an intensity ratio of 1:3:3:1 by the three neighboring CH3 protons. In principle, the two CH2 protons would also be split again into a doublet to form a doublet of quartets by the hydroxyl proton, but intermolecular exchange of the acidic hydroxyl proton often results in a loss of coupling information. Coupling to any spin nuclei such as phosphorus-31 or fluorine-19 works in this fashion (although the magnitudes of the coupling constants may be very different). But the splitting patterns differ from those described above for nuclei with spin greater than because the spin quantum number has more than two possible values. For instance, coupling to deuterium (a spin 1 nucleus) splits the signal into a 1:1:1 triplet because the spin 1 has three spin states. Similarly, a spin 3/2 nucleus splits a signal into a 1:1:1:1 quartet and so on. Coupling combined with the chemical shift (and the integration for protons) tells us not only about the chemical environment of the nuclei, but also the number of neighboring NMR active nuclei within the molecule. In more complex spectra with multiple peaks at
similar chemical shifts or in spectra of nuclei other than hydrogen, coupling is often the only way to distinguish different nuclei. A) Second-order (or strong) coupling The above description assumes that the coupling constant is small in comparison with the difference in NMR frequencies between the inequivalent spins. If the shift separation decreases (or the coupling strength increases), the multiplet intensity patterns are first distorted, and then become more complex and less easily analyzed (especially if more than two spins are involved). Intensification of some peaks in a multiplet is achieved at the expense of the remainder, which sometimes almost disappear in the background noise, although the integrated area under the peaks remains constant. In most high- field NMR, however, the distortions are usually modest and the characteristic distortions (roofing) can in fact help to identify related peaks. Second-order effects decrease as the frequency difference between multiplets increases, so that high- field (i.e. high- frequency) NMR spectra display less distortion than lower frequency spectra. Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically operating at frequencies at 200 MHz or above. B) Magnetic inequivalence More subtle effects can occur if chemically equivalent spins (i.e. nuclei related by symmetry and so having the same NMR frequency) have different coupling relationships to external spins. Spins that are chemically equivalent but are not indistinguishable (based on their coupling relationships) are termed magnetically inequivalent. For example, the 4 H sites of 1,2-dichlorobenzene divide into two chemically equivalent pairs by symmetry, but an individual member of one of the pairs has different couplings to the spins making up the other pair. Magnetic inequivalence leads to highly complex spectra which can only be analyzed by computational modeling. Such effects are more common in NMR spectra of aromatic and other non-flexible systems, while rapid rotation about CC bonds in flexible molecules ensures that the couplings between protons on adjacent carbons are identical, avoiding problems with magnetic inequivalence.
3.7 CORRELATION SPECTROSCOPY

Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy. This type of NMR experiment is best known by its acronym, COSY. Other types of two-dimensional NMR include J-spectroscopy, exchange spectroscopy (EXSY), Nuclear Overhauser effect spectroscopy (NOESY), total correlation spectroscopy (TOCSY) and heteronuclear correlation experiments, such as HSQC, HMQC, and HMBC. Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR. The first two-dimensional experiment, COSY, was proposed by Jean Jeener, a professor at Universit Libre de
Bruxelles, in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976.
23.8 SOLID-STATE NUCLEAR MAGNETIC RESONANCE

A variety of physical circumstances does not allow molecules to be studied in solution, and at the same time not by other spectroscopic techniques to an atomic level, either. In solid-phase media, such as crystals, microcrystalline powders, gels, anisotropic solutions, etc., it is in particular the dipolar coupling and chemical shift anisotropy that become dominant to the behaviour of the nuclear spin systems. In conventional solution-state NMR spectroscopy, these additional interactions would lead to a significant broadening of spectral lines. A variety of techniques allows to establish high-resolution conditions, that can, at least for 13C spectra, be comparable to solution-state NMR spectra. Two important concepts for high-resolution solid-state NMR spectroscopy are the limitation of possible molecular orientation by sample orientation, and the reduction of anisotropic nuclear magnetic interactions by sample spinning. Of the latter approach, fast spinning around the magic angle is a very prominent method, when the system comprises spin 1/2 nuclei. A number of intermediate techniques, with samples of partial alignment or reduced mobility, is currently being used in NMR spectroscopy. Applications in which solid-state NMR effects occur are often related to structure investigations on membrane proteins, protein fibrils or all kinds of polymers, and chemical analysis in inorganic chemistry, but also include "exotic" applications like the plant leaves and fuel cells.
23.9 APPLICATIONS
23.9.1 NMR spectroscopy applied to proteins Much of the recent innovation within NMR spectroscopy has been within the field of protein NMR, which has become a very important technique in structural biology. One common goal of these investigations is to obtain high resolution 3-dimensional structures of the protein, similar to what can be achieved by X-ray crystallography. In contrast to Xray crystallography, NMR is primarily limited to relatively small proteins, usually smaller than 25 kDa, though technical advances allow ever larger structures to be solved. NMR spectroscopy is often the only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins. Proteins are orders of magnitude larger than the small organic molecules discussed earlier in this article, but the same NMR theory applies. Because of the increased number of each element present in the molecule, the basic 1D spectra become crowded with overlapping signals to an extent where analysis is impossible. Therefore, multidimensional (2, 3 or 4D) experiments have been devised to deal with this problem. To facilitate these experiments, it is desirable to isotopically label the protein with 13C and 15N because the predominant naturally-occurring isotope 12C is not NMR-active,
whereas the nuclear quadrupole moment of the predominant naturally-occurring 14N isotope prevents high resolution information to be obtained from this nitrogen isotope. The most important method used for structure determination of proteins utilizes NOE experiments to measure distances between pairs of atoms within the molecule. Subsequently, the obtained distances are used to generate a 3D structure of the molecule using a computer program. 23.9.2 Medicine The use of nuclear magnetic resonance best known to the general public is in magnetic resonance imaging for medical diagnosis, however, it is also widely used in chemical studies, notably in NMR spectroscopy such as proton NMR and carbon-13 NMR. These studies are possible because nuclei are surrounded by orbiting electrons, which are also spinning charged particles such as magnets and, so, will partially shield the nuclei. The amount of shielding depends on the exact local environment. For example, hydrogen bonded to oxygen will be shielded differently than hydrogen bonded to a carbon atom. In addition, two hydrogen nuclei can interact via a process known as spin-spin coupling, if they are on the same molecule, which will split the lines of the spectra in a recognizable way. 23.9.3 Chemistry By studying the peaks of nuclear magnetic resonance spectra, skilled chemists can determine the structure of many compounds. It can be a very selective technique, distinguishing among many atoms within a molecule or collection of molecules of the same type but which differ only in terms of their local chemical environment. By studying T2* information a chemist can determine the identity of a compound by comparing the observed nuclear precession frequencies to known frequencies. Further structural data can be elucidated by observing spin-spin coupling, a process by which the precession frequency of a nucleus can be influenced by the magnetization transfer from nearby nuclei. T2 information can give information about dynamics and molecular motion. Because the nuclear magnetic resonance timescale is rather slow, compared to other spectroscopic methods, changing the temperature of a T2* experiment can also give information about fast reactions, such as the Cope rearrangement or about structural dynamics, such as ring- flipping in cyclohexane. An example of nuclear magnetic resonance being used in the determination of a structure is that of buckminsterfullerene. This now famous form of carbon has 60 carbon atoms forming a sphere. The carbon atoms are all in identical environments and so should see the same internal H field. Unfortunately, buckminsterfullerene contains no hydrogen and so 13C nuclear magnetic resonance has to be used. 13C spectra require longer acquisition
times since carbon-13 is not the common isotope of carbon (unlike hydrogen, where 1H is the common isotope). However, in 1990 the spectrum was obtained by R. Taylor and co-workers at the University of Sussex and was found to contain a single peak, confirming the unusual structure of C60.[2] 23.9.4 Non-destructive testing: Nuclear magnetic resonance is extremely useful for analyzing samples non-destructively. Radio waves and static magnetic fields easily penetrate many types of matter and anything that is not inherently ferromagnetic. For example, various expensive biological samples, such as nucleic acids, including RNA and DNA, or proteins, can be studied using nuclear magnetic resonance for weeks or months before using destructive biochemical experiments. This also makes nuclear magnetic resonance a good choice for analyzing dangerous samples. 23.9.5 Data acquisition in the petroleum industry: Another use for nuclear magnetic resonance is data acquisition in the petroleum industry for petroleum and natural gas exploration and recovery. A borehole is drilled into rock and sedimentary strata into which nuclear magnetic resonance logging equipment is lowered. Nuclear magnetic resonance analysis of these boreholes is used to measure rock porosity, estimate permeability from pore size distribution and identify pore fluids (water, oil and gas). These instruments are typically low field NMR spectrometers. 23.9.6 Process control: NMR has now entered the arena of real-time process control and process optimization in oil refineries and petrochemical plants. Two different types of NMR analysis are utilized to provide real time analysis of feeds and products in order to control and optimize unit operations. Time-domain NMR (TD-NMR) spectrometers operating at low field (2-20 MHz for 1H) yield free induction decay data that can be used to determine absolute hydrogen content values, rheological information, and component composition. These spectrometers are used in mining, polymer production, cosmetics and food manufacturing as well as coal analysis. High resolution FT-NMR spectrometers operating in the 60 MHz range with shielded permanent magnet systems yield high resolution 1H NMR spectra of refinery and petrochemical streams. The variation observed in these spectra with changing physical and chemical properties is modelled utilizing chemo metrics to yield predictions on unknown samples. The prediction results are provided to control systems via analogue or digital outputs from the spectrometer. 23.9.7 Earth's field NMR: In the Earth's magnetic field, NMR frequencies are in the audio frequency range. EFNMR is typically stimulated by applying a relatively strong dc magnetic field pulse to the sample and, following the pulse, analyzing the resulting low frequency alternating magnetic field that occurs in the earth's magnetic field due to free induction decay (FID). These effects are exploited in some types of magnetometers, and in EFNMR spectrometers. Their inexpensive portable nature makes these spectrometers valuable for field use and for teaching.
23.9.8 Magnetometers: Various magnetometers use NMR effects to measure magnetic fields, including Proton precession magnetometers (PPM) and Overhauser magnetometers. See also Earth's field NMR.
23.10 LET US SUM UP

When the nucleus is in a magnetic field, the initial populations of the energy levels are determined by thermodynamics, as described by the Boltzmann distribution Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy.

Elaborate as the NMR spectroscopic techniques.

Make a review of applications of NMR spectroscopy in Variety of Fields.
23.13 REFERENCES/
(1) G. A. Webb Nuclear Magnetic Resonance
(2) F. A. Rushworth, Nuclear Magnetic Resonance David Prestwich Tunstall (3) http://en.wikipedia.org/wiki/NMR_spectroscopy
LESSON 24- CIRCULAR DICHROISM SPECTROSCOPY

Contents 24.0 Aims and objectives 24.1 Introduction 24.2 Physics of CD and ORD 24.3 CD data analysis 24.4 Application to biological molecules 24.5 Limitations 24.6 Lesson End Activities 24.7 Check Your Progress 24.8 References

In this lesson we have described about circular dichroism spectroscopy, its application to biomolecules and limitations. After reading this lesson you will be able to: Know the concept of CD and ORD Describe CD data analysis Applications of CD spectroscopy
24.1 INTRODUCTION
Circular Dichroism (CD) is observed when optically active matter absorbs left and right hand circular polarized light slightly differently. It is measured with a CD spectropolarimeter, which is relatively expensive (~$70k). The instrument needs to be able to measure accurately in the far UV at wavelengths down to 190-170 nm. In addition, the difference in left and right handed absorbance A(l)- A(r) is very small (usually in the range of 0.0001) corresponding to an ellipticity of a few 1/100th of a degree. The CD is a function of wavelength. CD spectra for distinct types of secondary structure present in peptides, proteins and nucleic acids are different. The analysis of CD spectra can therefore yield valuable information about secondary structure of biological macromolecules.
Fig91: The CD Spectrometer
24.2 PHYSICS OF CD AND ORD

Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase. A projection of the combined amplitudes perpendicular to the propagation direction thus yields a line.When this light passes through an optically active sample with a different absorbance A for the two components, the amplitude of the stronger absorbed component will smaller than that of the less absorbed component. The consequence is that a projection of the resulting amplitude now yields an ellipse instead of the usual line (draw on a sheet of paper and check). Note that the polarization direction has not changed. The occurrence of ellipticity is called Circular Dichroism - it is not the same as optical rotation. Rotation of the polarization plane (or the axes of the dichroic ellipse) by a small angle a occurs when the phases for the 2 circular components become different, which requires a difference in the refractive index n. This effect is called circular birefringence. It can be shown that when CD exists, optical rotation must exist as well, and they are directly related by a Kronig-Kramers transformation (see anomalous x-ray dispersion (scattering factors f ' and f '') for more on K-K). The change of optical rotation with wavelength is called optical rotary dispersion, ORD.
Fig 9 2 : (a) Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase. (b): different absorption of the left- and right hand polarized component leads to ellipticity (CD) and optical rotation (OR). The actual effect is minute and using actual numbers the ellipse would still resemble a line.
24.3 CD DATA ANALYSIS
As mentioned in the introduction, the difference in absorption to be measured is very small. The differential absorption is usually a few 1/100ths to a few 1/10th of a percent, but it can be determined quite accurately. The raw data plotted on the chart recorder represent the ellipticity of the sample in radians
which can be easily converted into degrees
To be able to compare these ellipticity values we need to convert into a normalized value. The unit most commonly used in protein and peptide work is the mean molar ellipticity per residue. We need to consider path length l, concentration c , molecular weight M and number of residues
in proper units (CD spectroscopists use decimol)
This finally reduces to
The values for mean molar ellipticity per residue are usually in the 10.000's
24.4 APPLICATION TO BIOLOGICAL MOLECULES

In general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of the dextrorotary (e.g. some sugars) and levorotary (e.g. some amino acids) molecules they contain. Even more important is that a secondary structure will also impart a distinct CD to its respective molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures. CD is closely related to the optical rotatory dispersion (ORD) technique, and is generally considered to be more advanced. CD is measured in or near the absorption bands of the molecule of interest, while ORD can be measured far from these bands. In principle these two spectral measurements can be interconverted through an integral transform, if all the absorptions are included in the measurements.
The far-UV (ultraviolet) CD spectrum of proteins can reveal important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha- helix conformation, the beta-sheet conformation, the betaturn conformation, or some other (e.g. random coil) conformation. These fractional assignments place important constraints on the possible secondary conformations that the protein can be in. CD cannot, in general, say where the alpha helices that are detected are located within the molecule or even completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents, e.g. Guanidinium hydrochloride or urea. In this way it can reveal important thermodynamic information (such as the enthalpy and Gibbs free energy of denaturation) about the molecule that cannot otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to alpha-helix fraction estimation. The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure. The signals obtained in the 250-300 nm region are due to the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine, tyrosine, cysteine (or S-S disulphide bridges) and tryptophan amino acids. Unlike in far-UV CD, the near-UV CD spectrum cannot be assigned to any particular 3D structure. Near-UV CD spectra can also provide structural information on the nature of the prosthetic groups in proteins, such as heme groups e.g. in hemoglobin and cytochrome c. CD gives less specific structural information than e.g. X-ray crystallography or protein NMR spectroscopy, that both give atomic resolution data. However CD spectroscopy is a quick method, that does not require large amounts of proteins and extensive data processing. Thus CD can be used to survey a large number of solvent conditions, varying temperature, pH, salinity and presence of various cofactors. CD spectroscopy is usually used to study proteins in solution, and thus it complements methods that study the solid state. This is also a limitation, in that many proteins are embedded in membranes in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films.
24.5 EXPERIMENTAL LIMITATIONS

CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100-200 nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured.
Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range where structural features exhibit differential absorption of circularly polarized light. Phosphate, sulfate, carbonate, and acetate buffers are generally incompatible with CD unless made extremely dilute e.g. in the 10-50 mM range. The TRIS buffer system should be completely avoided when performing far-UV CD. Borate and ammonium salts are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the far UV, 0.1 mm path lengths are not uncommon in this work. In addition to measuring in aqueous systems, CD, particularly far-UV CD, can be measured in organic solvents e.g. ethanol, methanol,trifluoroethanol (or TFE). The latter has the advantage to induce structure formation of proteins, inducing beta-sheets in some and alpha helices in others, which they would not show under normal aqueous conditions. Most common organic solvents such as acetonitrile, THF, chloroform, dichloromethane are however, incompatible with far-UV CD. It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the to * orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen- free instrument (filled with pure nitrogen gas). Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 nm is to design the rest of the optical system to have low losses in this region. Critical in this regard is the use of aluminized mirrors whose coatings have been optimized for low loss in this region of the spectrum. The usual light source in these instruments is a high pressure, short-arc xenon lamp. Ordinary xenon arc lamps are unsuitable for use in the low UV. Instead specially constructed lamps with envelopes made from high-purity synthetic fused silica must be used. Light from synchrotron sources has a much higher flux at short wavelengths, and has been used to record CD down to 160 nm. At the quantum mechanical level, the information content of circular dichroism and optical rotation are identical.
24.6 LET US SUM UP

In this unit, we have discussed, CD and ORD spectroscopic concepts Had a detailed study on CD data analysis
Applications of CD spectroscopy.

Compare and contrast CD and DRD Spectroscopy.

Make a comparative study on IR, UV, NMR and CD spectroscopic techniques and give your overview on all (Refer Unit -5 Lesson 1 to 4).
24.9 REFERENCES
Introduction to Biophysics; Vasantha Pattabiraman, N.Gautham NMR Spectroscopy. 2nd Ed.; H. Gunther, John Wiley and Sons, Chichester, 1995. Nuclear Magnetic Resonance; P.J. Hore, Oxford University Press, Oxford, 1995.

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UNIT - I LESSON 1 THEORY OF ATOMIC AND MOLECULAR ORBITALS

1.0 AIMS AND OBJECTIVES

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Fig 1: Discrete solutions of wave equations.

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Fig 3: Energy levels in a molecule

1.2 THEORY OF ATOMIC ORBITAL

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1.3 LET US SUM UP

1.4 LESSON END ACTIVITIES

1.5 CHECK YOUR PROGRESS

W.L. Jolly Charles M. Quinn

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LESSON: 2 MOLECULAR ORBITAL THEORY

2.0 AIMS AND OBJECTIVES

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2.2 FORMING MOLECULAR ORBITALS

Fig 4: Formation of molecular orbitals of H2 molecule.

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Fig 5: Energy level of an H2 molecule

Fig 6 The status of the electrons of hydrogen atoms

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Fig 7: Formation of Sigma orbitals

Fig 8: Formation of Pi orbitals

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Fig 9 The models describing O2 and F2

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Fig 10 The models described for B2 , C2 , N2

2.3 BOND ORDER

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2.4 ORBITALS FOR SELECTED MOLECULES

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Fig 14 : A pictorial representation of the energy diagram for Benzene

Fig 15 The position of electrons in a benzene ring

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2.5 LET US SUM UP

2.6 POINTS FOR DISCUSSION

2.7 CHECK YOUR PROGRESS

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L E S S O N 3 - LINEAR COMBINATION OF ATOMIC ORBITALS AND VALENCE BOND THEORY

3.0 AIMS AND OBJECTIVES

3.1 LINEAR COMBINATION OF ATOMIC ORBITALS

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Fig 16 : Representation of bonding in water molecule

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3.2 VALENCE BOND THEORY

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3.3 LET US SUM UP

3.4 LESSON END ACTIVITIES

(2) http://en.wikipedia.org/wiki/Valence_bond_theory (3) http://www.iupac.org/goldbook/VT07125.pdf

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LESSON 4 - RESONANCE STRUCTURES

4.0 AIMS AND OBJECTIVES

4.2 DRAWING RESONANCE STRUCTURES

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Fig 17 : Lewis structure of CHO2 -