Biochemistry Lecture 1

The BIG picture:

1. Interconnections
2. Function and Form
3. Classification based on energy sources
4. The cell compare and contrast between different types
5. Structural Hierarchy
What is Biochemistry? CH 1 pg 1-12

Repair,

Systems depend on ea. Other

High degree of chemical complexity combined with a basically rigid microscopic hierarchy or organization

Systems for extracting, transforming, and using energy from the environment
o Mechanical Transformation
o Chemical Transformation
Most estract/ use energy
Lowest state = most steady, stable equilibrium
Decay Molecular state of disorder surrounding environment

Precise Self-replication/self assembly

Sensory Preception
Force change or adaption biochemical changes expression/regulation
Moths recessive black gene becomes dominant
Those who adapt to changes or have the ability to adjust internal chemical processes in response to a
environmental perception based on gauging that change, have a better process of retaining
functionality/survival.
Defined Function Everything has (or did have) a biological or biochemical purpose
Alpha-fetoprotein protein enzyme present in the fetus
Rarely present in developmental human, but is expressed specifically in certain forms of cancer
o This is a biological marker for pathology (tumor marker)
Regulation, interaction, not an individual part all work together to maintain life
Understanding the chemical function we can explain how cellular process work, or how they can go
wrong
Record of events and developments Evolution (life history)

Enzyme in different species, shows

evolution, but similarities

Diversity of Life as a reflection of the diversity of biological

reactions
Common elements are involved; understanding these
common elements, the mechanism of how they interact or how they are regulated is important and has
far reaching implications..
Understanding the basic principles of biochemistry, we ultimately have a better understanding of the
complexity that chemistry plays in providing a platform for life

From a single cell or to a whole plant or person, understanding these mechanisms have a major
importance in medicine, biology, food production, agriculture, etc
Basics: Biochemistry 101 pg. 3
The cell: a unit
Common features:
o Plasma Membrane: Physical barrier, doors/controlled openings active transport mechanisms,
ion concentration gradients etc trans-membrane proteins, ion channels (highly selective
membrane bound proteins that provide a selective entrance into a cell)
o Cytoplasm: materials encased by the plasma membrane
o Contents within the cytosol: cellular matrix, not free flowing, highly organized and partitions,
contains a host of molecules/organelles; by products, metabolites; intermediate compounds
o Nucleus/nucleoid (bacteria) houses the genetics of the cell (DNA)
Distinct between organisms Diversification
-Bacteria structure nucleoid, not separated
-Higher organisms nuclear envelope encases genetic information (double membrane)
o Eukaryotes: A unicellular or multi-cellular organism with cells having membrane bound nucleus,
multiple chromosomes, and internal organelles
o Prokaryotes: A bacterium; a unicellular organism with a single chromosome, no nuclear
envelope, no membrane bound organelles
Cellular limitations all based on size
Without active process of diffusion, or structural organization to gain/remove metabolites/wastes, a cell
is then limited by its ability to survive based on the ability of
diffusion within an aqueous system/cytosol.
L- Healthy Lungs
Surface to volume ratio balance between ability to exchange with
R- Smoker
its surrounding environment
pH change affects many
Ultimately limits cellular growth/size
things in the body

Classification: All living things fall into 3 Domains of Life pg.4

KscA K+
Channel

Classification of Organisms based on source of energy and carbon pg. 4

Eukaryote Cells Animal v. Plant pg. 6

Separation of Eukaryotic Organelles

Differential centrifugation separate density by rotating at high speed
Sucrose gradient centrifugation

Liver Cells

Freezing & Pillowing technique

Eukaryotic Cell Matrix Cytoskeleton pg. 8

Cellular Matrix in Dynamic

Moving/transporting corridors surface proteins
Foundation for specialized areas involved in biochemical reactions (provides a level of regulation no
good to have everything in a big soup)
More specifically actin and microtubules provide motion

Requires expenditure of energy

-Exocytosis: Allows secretion into the surrounding environment vesicle membrane becomes part of the
plasma membrane

-Endocytosis: Allows uptake from the surrounding environment vesicle membrane is identical to the plasma
membrane

Structural hiearcy in the molecular organization of cells pg. 10

Memorize 20 Amino Acid Names and Structure pg. 9

Lecture 2
Geometry and functionality of the Carbon Bond CH 1 pg 11-39

Biomolecules are compounds of carbon with a variety of functional groups

double & triple bond stores more energy

Combination and complexity of functionalities pg. 11-14

Macromolecules contain multiple-functionalities, these can include: (and further grouped in specific
chemical species)
o Alcohols (1 or >OH groups)
o Amine (Amino groups)
o Aldehydes (Carbonyl groups)
o Ketons (carbonyl groups)
o Carboxylic acids (carboxyl groups)
Has ability to store energy through making and breaking bonds
Common Carbon-Hydrogen based functionalities
Common Carbon-Oxygen based functionalities

-Phenyl very stable

Common Nitrogen-based functionalities

-Can convert to each other, ether found in fats

Common Sulfur-based functionalities

-Sulfhydryl disulfide
-Thioester on CoA, S&C=O

Common Phosphate-based functionalities

Can hydroxylate it, can tell whats breaking by looking at energy

(temp changes)
Read and understand the concept of Molecular mass/Daltons Box 1-1 pg.14

Dalton = atomic mass unit

Primary metabolites are decomposed to maximum, can use it for plants that biotransform the product
o Can be used for insectiside etc
Secondary: we can consume, but later metabolize waste

Macromolecules are the major constituents of cells

Biological molecules macromolecules; HMW polymers of simpler precursors

Simple sugars monosaccharides;

>40 AA = peptide

Understanding the importance of three-dimensional Configuration.conformation pg.16

Specific orientation/arrangement in 3D space
Stereo-isomers: same chemical bonds, different configuration different chemical and optical properties
Biochemical reactions are stereo-specific, they are selective to compounds
Configuration is conferred by presence of:
o Double carbon bonds (no rotation)
o Chiral centre

Across
Side
Geometric
isomers
cis-trans isomers

If there is once chiral atom in a molecule there will be two stereoisomers (entantiomeric) forms

Review

RS System assigning stereochemistry unambiguously pg. 18

Some chemical groups, in close proximately to each other (i.e. H) repel thus the ability of free rotation
via the carbon-carbon, this forms a staggered complex (lowest energy state, more stable)
Yet replacement of these functionalities, this will possibly change this state of configuration

pg. 19

Case study: The importance of stereochemistry and biochemistry Thalidomide

Originally used for morning sickness in pregnant women and as sleeping aid, not FDA approved
o One enantiomer (R) is effective against morning sickness
o Other enantiomer (S) is teratogenic during fetal development
Enantiomers are converted to each other in vivo if you give only one isomer, the bodies pH will
interconvert to form equal amounts of each enantiomer
Present use: leprosy and multiple myeloma in non-pregnant persons

Work = survival pg.21

Isolated system = matter/E not exchanged with surroundings stagnant (jar of sugar not reacting)
Closed system = ONLY E exchanged (heating sugar = melting)
Open system = Both Matter/E exchanged (adding solution to suger = dissolve, energy transfer, or yeast
CO2)

Living organisms exist in a dynamic steady state, NOT at equilibrium with their
surroundings

The Sun without it, were dead

THERMODYNAMICS: The roles of Entropy (S), Enthalpy (H), and free energy content (G): Supply of E for
the synthesis of macromolecules pg.23
2nd Law of Thermodynamics: tendency to move towards an ever greater state of disorder
Yet the TOTAL entropy is continually increasing
o The breakdown trickle effect: to become its smallest most stable form
Chemical reactions is a closed system:

Thus holding the reaction at constant T (Kelvin) G is determined by H reflects formation/breaking of

both covalent and noncovalent interactions
G = H- T S (-G = release energy, +G = uses energy)
o S=Change in systems randomness, is positive

-G drives +G by coupling
-sum/net energy = (-)
ATP = the currency of life

Can do this 3x AMP, ADP, ADP

AMP secondary messenger system, not used as much

[Energy, structure and G requirements]

The transformation of energy in living systems bioenergetics pg.24

If youre going the opposite way, release energy, needs enzymes; same energy to break it and make it

Heat = loss of energy

Understanding the concepts of G and Keq pg.25
aA + bB cD + dD
Keq=Concentration of reactants / products

Lowering the energy barrier Enzymes pg. 27

Enzymes:
o Highly substrate specific (chirality, energy boundaries)
o Highly regulated, can consume a lot of energy and material, as well as produce a lot energy

o
o
o
o
o
o
o
o
o
o
o

1,000s of different enzymes required in each cell

Stereoisomer selective
Biocatalysts
Proteins (99%)
Bind during transition state (exergonic) may require a little G specifically to the enzyme
Upon binding, energy is released, lowers activation energy/energy barrier
Greatly increases rate of reaction
Some require co-factors (metals, other proteins)
Enzymes are not consumed during reaction, only recycled
Enzymes play an essential role in consecutive complex reaction pathways
90% of diseases are reflected by enzyme malfunction/mutation etc

amount of substrate can

affect enzyme and level of product
Positive/energy feedback for control
Replication = survival pg. 29-32
DNA Deoxyribonucleic acid

Passing on information for survival

Can pass on mutation

Evolution history of survival

Genetic analysis provides a pedigree of the various changes that have occurred throughout time

Mutation as an advantage, a mutation in protein can affect higher level substrates

Genetic analysis provides a pedigree of various changes that have occurred throughout time
Provides opportunity for genetic counseling
Chemical Evolution pg. 33

produce starting materials to life

Eukaryotic Evolution prokaryotes pg. 37

cyanobacterial component

PPT 3: Water the essence of life

Weak inter- and intra-molecular interactions in biochemistry, water can play as chemical, buffer
Polarity of a molecular dictates how it interacts in a aqueous environment
o Polar biomolecules: dissolve (H2O/H2O interactions are placed with energetically favorable
H2O- solute interactions) has ()delta G
o Nonpolar biomolecules ppt. or cluster (unable to participate in H2O/H2O interactions) has
(+)delta G

Together strong
individually weak
Unique chemical and internal cohesion properties of H2O provides usual characteristics
o Higher melting point boiling point
o Heat vaporizations
Why does ice float? Bond orientation and crystallization makes different physical property and density

Hydrogen bonding acts as layer/cushion and maximize interaction

Ice: Intra-molecular interactions = 4H2O
Makes it less dense why ice floats
Highly organized and structured lattice
To break bonds of ice:
o H20 (s)H20 (l) H = +5.9KJ/mol
o H20 (l)H20 (g) H = +44.0KJ/mol
Melting:
G = H-T
o G driving force
o H making/breaking bonds (+ve for melting)
o S- randomness ( entropy
G = -ve spontaneous less organize/random
o (s) < (l) << (g)
Hydrogen bonds play important roles in biomolecules, yet each has different strengths, diff G

H-H strongest with orientation which maximized inter/intra molecular electrostatic interactions

allows to stabilize many things (DNA&Base Pair RNA)

Effect of solevent interaction upon solute ions
Hydrophillic: compounds that dissolve easily in polar solvent (water)
Hydrophobic: compounds that dissolve in nonpolar solvents (ether)

Water screens electrostatic interactions between solute molecules hydrates ions

process of salt dissolving. Increase charge distribution

Nonpolar compounds force energetically unfavorable charges in the structure of H2O

Amphiphatic: contains both polar and non-polar identities, when mix in H2O hydrophilic regions tend to
dissolve, but the non-polar hydrophobic region advoids contact with H2O micelle formation
Fatty Acid:

Buffer alkyl chain of fatty acid

More heavily interactive

Likes attract, will try to sqeeze out water, clusters of lipid form, entropy increase
More fatty acid = Micelle

shell that cluster around hydrophobic region inside

Forces that hold nonpolar regions together hydrophobic interactions
Greatest thermodynamic stabilized structure achieved by the maximal exclusion of H20 within its
hydrophobic region
Hydrophobic interactions among lipids, lipids and proteins are most important determinants is structural
membranes Hydrophobic interactions between nonpolar Amino Acids stabilize 3D structure of proteins and
protein-protein interactions

Water pushed out by adding energy, decreases deltaG

allows to stabilize molecule

Solutes alter chemical properties of solvents:

glucose has no ions to dissociate

Depending on the number of particles (moremore colligative properties)

water plays important role in salvation of protein structure, w/o it

can affect binding sites

Molecular water filling in gaps

Osmolarity
H2O moves from high [H2O] to lower H2O]
In the presence of semipermeable membrane, water freely difuses, but not the solutes
osmotic pressure (II) measured force to resist water movements

ic = similarity i; vant Hoff factor reflection to how far a solute will dissociate
c; concentration of the solute
Thus NaCl solution complete dissociation to Na+ and Cl+ number of solute particles (i=2)
So II = sum of contributing solutes in dissociated form i.e. = RT (i1c1 + i2c2 +.incn)

Osmosis: H2O movement across semipermeable membranes is driven by in osmotic pressure

Essential mechanism in cellular life
o Solution of equal osmolarity = isotonic (cells neither gains or losses H2O)
o Solution with higher osmolarity than cytosol = hypertonic (cell shrinks with loss of H2O)
o Solution with lower osmolarity than cytosol = hypotonic (cell swells with gain of H2O)

-Water contamination adding sugar can affect osmolarity of bacteria and make it safe to drink
Osmolarity is dependent on number of dissolved particles not mass. Thus macromolecules have less effect then
osmolarity of their individual componenets
Polysaccharide storage
o In the form of starch/glycogen contributes less II, than if in the form of glucose
o Plants use II to provide cellular and structural rigidity or even movement as s in turgor pressure
resulting in osmotic pressure on the cell wall
o Isolation of osmotic sensitive cellular organelles isolation must be carried out in isotonic
solution to prevent cells rupturing ex: brain material gray matter
o Lysis cell via exposing them to hypotonic solution

Importance of H+ ions in Biochemistry

Ionization Equilibrium constant
pH
Weak acid/bases: change functionality by protination and deprotination
Titration curves pKa
Biological buffers (neutralizing very acidic gastric fluid)
Henderson-hasselback equation
Biochemical and mechanical implications of pH
Water as a reactant
pH and Temp

-Temperature changes ionization and changes pH

Pure wate is slightly ionizable described by Equilibrium Constant (Keq)

Changing only OH- or H+ affects Keq,

Affects stability of molecule
Measuring voltage of water in its purist form
Starts to have changes when solutes are added
Solutes can combat the reaction

pH scale assigns H+ and OH- concentrations

pH = log 1/[H+] = - log [H+]
Thus for a Neutral solution
o [H+] = [OH-]
o [H+] = 1.0x 10-7 M
o pH = log 1/ 1.0x 10-7 M
= log 1.0 + log 107
= 0+7
= 7 reference for Neutral pH

pH+pOH=14

log relationship to ion concentration

Hunger: increase proton in blood decrease pH

Weak acids and bases have characteristic dissociation constants (Ka)
Focus on the use of weak acids/bases those that do not complete dissociation in solution (ex having
buffer abilities)
Conjugate acid-base pair consistant of a proton donor and proton acceptor
o CH3COOH H+ + CH3COOH+ Donor
H+ acceptor

Titration Curves reveal the pKa of Weak Acids

Measuring resulting pH against the amount of NaOH (molar equivalents) added

Monoprotonoic acidreleases one H

Buffer release of hydroxyl or H+ ions to reach and maintain a certain pH
Living cells should be ideally pH7

Buffers are mixtures of weak acids and their bases Resisth changes in pH with addition of H+ or OHInterlinkage of two reversivle processes of:
Ionization
Dissociation
Governed by Kw and Ka

Each conjugate acid-base pair have characteristics of buffer zone

Henderson-Hasselbalch Equation *exam

Combating pH changes in Cells and tissues

Intracellular/extracellular fluids constant pH
pH combated by proteins via their amino acids
Amino acids contain ionizable functional groups that are weak acids or weak base

Proteins that contain Histidine (pKa=6) can act as a buffer in near neutral pHs
This becomes a very important interactive feature in biomolecules (proteins/peptides) and their functions and
structure

Multiple sites for deprotination

H2O as a reactant in biological reaction
Not just a solvent but an essential participant in biological reactions as proton donor
Condensation reaction H2O is eliminated while the opposite of this same reaction, the production of
H2O is hydrolysis reaction

Hydrolysis reaction, responsible for enzymatic breakdown of

proteins, carbohydrates, and nucleic acids, into the subunit forms (amino acids, sugars, nucleotides)
Family of enzymes = hydrolases
ACh-Neurotransmitter
Other examples:

Amino acids, Peptides and Proteins are the most abundant material in cells
Proteins are the molecular consequences of genetic information
Late 1990s saw the boom in gene technology, this will now be transferred to biochemistry/protein
biotechnology
20 AMINO ACID Structure, full name, three letter code, one letter code

alpha carbon chiral carbon

R chain differs by size, hydrophobicity, chanrge, structure, how it reacts with other side chains
How we represent amino acids
L-life, D- death: we do not produce D-amino acids
Absolute configuration: L- and D- system in amino acids
a-carbon chiral centre
Enzymes change D to L amino
acid

L/D Rotation in regards to fractionary light

Different Representations of Amino Acids
Molecular Hierarchy

-Condensation reaction reduce molecular water by consuming an breaking bonds

-Tags on protein to indicate where they move to in the cell, can be suicide pill as well
-Subunits of proteins come together to form ionic homo or hetro dimer

Glycine
(R=H)only AA
thats
non
chiral,
Smallest
molecula

Central to understanding amino acid is their R group properties in biological pH

non-polar/hydrophobic (water fearing)
o Phe, Tyrz(aromatic), Trp, have side chains
highly polar/hydrophilic (water loving)

Asparagine & Glutamine is the nitrogenous form of Asparatate and Glutamate (Carboxylic form)
Cysteine SH S- , produces thiolate

Naming System

(Sulfur Containing) can be oxidized to sulfoxide, is a problem

Nonpolar, hydrophobic, hydrophobic interactions
Soluble in H20/hydrophilic,
structure stabilizing characteristics
H-H bond formation, hydroxyl, sulfhydryl
and aminde groups

Cysteines ability to form covalent bonds with other cysteine moieties

Can form dimer in pka 8, by oxidation

Key elements in structural stabilization:
intra-disulfide bond formation within the sequence
inter-disulfide bond formation between individual molecules

Phe and Try not happy in water

Hydrophobic in nature
Participate in hydrophobic interactions
Tyr forms H-H bond via hydroxyl group

UV Light
Tyr and Trp used commonly for [protein] determination
Labert-Beers law: Absx =Ex[C](l)

Histidines ability to ionize at physiological pH

Negatively
charged at
Physiological pH

Note the relations between Asp and Asn Glu and Gln

Phe Nonpolar
Leu
MetAla
Gly Ile
Charged
Cys
P
Glu Ly Tyr
Trp r
AsnGln
s
His Asp
Arg
o
SerThr
Polar
See http://www.elmhurst.edu/~chm/vchembook/561aminostructure.html
Polar side chains contain groups that are either charged at physiological pH or groups that are able to
participate in hydrogen bonding
An essential amino acid for an organism is an amino acid that cannot be synthesized by the organism
from other available resources and therefore must be supplied as part of the diet
These Ten Valuable Amino Acids Have long Preserved Life In Man Thr, Trp, Val, Arg, Ala, His. Leu Pro,
Lys, Ile, Met
Various on a theme post transitional modifications (PTMs)

Enxyme that makes hydroxyproline from proline is Vit.C dependent, if not present can cause scurvy

Use the Net Charge 0 to calculate PI value

The pKa is greatly affected by the surrounding chemical environment thus some pKas are very different to
what would be normally expected chemically

Multiple ionizable R groups provide distinct tritation curves

The essence of peptide, polypeptide, and proteins the peptide bond

4 Peptide bonds, 5 AA
Change shape to stabilize

A peptide/protein contains only one free alpha amino end and one free alpha carboxyl group at opposite
ends of the amino acid chain
The combination of the amino-function to carboxyl function, forming the peptide back bond (peptide
bond) is a non-ionizable constitute in its covalent form
Some peptides contain a PTM N- or C- terminal that will mask the ability of the functional group to
ionize
R groups will have the ability to ionize in a characteristic manner
Combination of these ionization characteristics within a individual sequence/ R groups have predictive
nature that is reflective of the combination of amino acids present
Importantly is the unique combination of the amino acids that give rise to structure; it is the structure in
combination with specific R groups that give rise to functionality Unique combination of amino acid give
rise to chemical features like ionization at specific pH (aka pIs, hydrophobicity, amino acid compositional
nature, etc)
-Proteins coming in various sizes and subunit compositions

Multi-subunits: noncovalent associated

proteins (identical or different)
Oligomeric if its 2 or more subunits that
are identical
Protomers identical subunits associated
in multiple forms alpha subunits/ beta
subunits dimmers (2); tetramers (4)

Calculation of the number of approximate amino acids: MW/110*

*Average MW of predominant amino acids
Protein (Pure) peptide cleavage, acid/base hydrolysis 00>
o Mixed of free amino acids
o Characteristics and proportional representation in each different protein
o Typical AA do not occur in equal amounts informative
UUpon acid hydrolysis, certain AA degradation to form
byproducts, some amino acids become indistinguishable
Asp&Asn (Asx); Glu&Gln (Glx)
Some proteins combination additional chemical
functionalities protein conjugates

Structural relationship
AA-Most important and influential give rise to secondary,
then tertiary and quatenary
Some AA people cant metabolize
Phenylpyruvate produced as a response to overproduction of
phenylalanine
Effects ketone bodies, pH levels
Protein, Peptide,Isolation and Biochemical characterization techniques and methology

Size Exclusion
Chromatography
Isocratic run- one solvent
separate based on
molecular size Large
molecules come out first,
have least resistance,
small molecules have
more resistance, take

Ion Exchange Chromatography/ Anion-Cation Exchange

Gradient run mixing
of two solvent at
proportions

Negarive charge attaract pos. molecules

Affinity Chromatography

Affinity Chromatography: More specific for compound of interest uses antibodies

Commonly use immobilized antibodies that have a specific ligand (antigen) affinity
Nickel NTA high affinity for Hist tag on the end of the protein of interest especially useful for
recombinant protein purification
High pressure liquid chromatography: extremely common in biochemical and pharmaceutical laboratories
Not very useful for protein separation, better suited to:
o Small organic molecules
o Peptides
o Polypeptides
Separation is based on how a peptide/polypeptide is soluble in an organic mobile phase
Peptide binds to stationary phase of carbon (silica)
Eluted with increase gradient of organic
When (organic) reaches the solubility of bound peptide peptide comes off
Highly reproducible and very robust

Protein migrates within a electric field based on total charge composition

SDS page electrophoresis

Isoelectric focusing 2D gel
electrophoresis

SDS Page Gel Electrophoresis

o Sodium dodecyl sulfate (SDS): detergent: allows proteins to move in the matrix based on mass (SDS
binds proportionally providing varying degrees of negativity based on mass)
o Larger the protein, more SDS binding, higher the Ve charge, greater retention, slower movement)
o Native Gels: protein is in native conformation
o Non-native Gels: protein structure is reduced to non-native form
Only good for
proteins, not good for
peptides, too small

More protein = deeper stain

densitometry

Determination of protein concentration by gel

dilute

Determination of protein concentration by gel densitometry

o Stain gel with charge binding colour stain (Coomassie blue)
o Measure the amount of stain retained
o Compare against STD curve
Can also do it by beer-lambert law

SDS-PAGE: Molecular mass analysis

pH&pI

pI:pH at which the net=0

o Reflective of all the composite amino acids together provides a key ability to separate closely related
proteins
o By changing the pH conditions, we effect the pI Method for separating proteins (re-examine ion
exchange chromatography)
o Pepsin stomach to break down proteins
2D Gel electrophoresis
o Provides better separation based on combination of MW and then pI (or vise versa)

Examine specific activity

(colorimetric, enzymatic) via assaytrace the isolation and purification
of the material
Specific activity: 1.0 unit enzyme

activity; transformation of 0.1Mol

of substrate at 25 o C

Protein digests

Know: Trypsin, chymotripsin, pepsin and cyanogens bromide

o Isolated/purified proteins wont work on crude mixtures
o Specifically enzymatic conditions required
o Some are highly specific to the alpha alpha sequence they are targeting
o Others non-selective and chew through the sequence
o Chemical cleavage agents highly specific chemical reaction that results in breaking of the peptide
backbone
Enzyme Cleavage

Has Lysine
C-terminal: no lysine
o Trypsin (K/R) = protease , cleaves at the C-terminal a-carboxypeptidase
o c.f. Pyro-glutaminase (Z or pyro-Glut/PTM cyclic form of Glu) which is a a-aminopeptidase
needs specialized enzyme
o c.f. aminopeptidase (non-specific for a-amino functions, cuts one aa of at a time from the N-terminus)

Understanding the amino acid composition

Doesnt work with cystiene

Protein Mapping

Obtain sequence overlap to determine the sequence. First need to determine if peptide/protein
has disulfide bonds

Disulfide reduction and derivitization

P100
Cysteic acid and Acetylated cysteine are PTH friendly

Additional Example (more detailed) Read & Understand

Polypeptide in oxidized form,
hydrodize it
Trypsin digest
Produce 3cuts = 4 peptides

Process to take to establish full sequence

The present revolution in Biochemistry- mass spectrometry
Electro Spray Mass Spectometry (ESMS)

charged state, can get precise mass

Ladder sequencing de nova sequencing

Solid Phase Peptide Synthesis (SPPS)

Bruce Merrifield won nobel prize for

Can make 100s of AA, build them up backwards

Adding additional amino acids to support matrix sequentially

Deprotect and remove Fmoc group

Cleavage from the support matrix

Importance of Quality control- achieving maximal yields for the correct target peptide
Sequence mining - bioinformatics

Present information based on evolution, function, structure

Start making searches for sequences with dominant structures, looking for sequence alignment, differences
between homology (homologous amino acids) and identity (physical property)
Proteins are not stagnant structures
3D structure determined by 1o by aa sequence Function arises from conformation
Proteins exists in a number of structurally stable forms via noncovalent interactions

Native state: lowest G energy

But what keeps this in its lowest state when only G 20-65 KJ/mol separates native from unfolded structure?

Conformational entropy unfolded has the highest state, more H2O interact via H-H bonding resulting
in maintaining of the unfolded state
In the native state these have to be counterbalanced by (i) disulfide bond formation, (ii) weak
noncovalent interactions: H-H bonds, Hydrophobic and ionic interacts
So numerous have a major contribution
Native state has the maximal number of weak interactions
o 200-460 KJ/Mol : Covalent bond
o 4-30 KJ/mol to dislodge weak interactions

Ion binding protein, comes close to ligand, binds the iron, and undergoes 3D change to get to stable structure
The Native Conformation of a protein is favored
H2O is key
H-H bonds via H2O presence surrounds hydrophobic molecule, there is optimal arrangement of
structured H2O (shell) salvation layer or effect

want to minimize salvation & h-bonding

Net G is derived from in weak interactions: in entropy resulting from hydrophobic core clustering
Stability in folded form
Proteins can be destabilized necessity for hydrophobic core cluster to be H-H bond
Significant difference between groups in their folded and unfolded state due to the type of interactions
that can undertake with their surroundings
Salt bridges: cooperative partnerships one form of bonding may facilitate another
Summary:
o Hydrophobic residues buried in protein core away from H2O
o H-H bonding is essential and needs to be at its maximal number and ability
Important: Depending on surrounding environment (hydrophobic v. hydrophilic), the rules differ
slightly, but weak interactions still play a dominant role
Understanding geometry of the peptide bond

The carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a
small electric dipole, virtually all peptide bonds in proteins occur in this trans configuration. This effects bond
length
Different bond length affect the ability of rotation

Regions of rigidity and areas of flexible peptide back bond

Heavy influence of side chains being partial pos or neg
Degree of flexability depends on R chain functionalities

C-alpha is the central position of flexibility

(phi) angle of rotation at N-Ca bonds

(psi) angle of rotation at Ca-C bonds

w tends to be planar

Limitations of and due to steric hindrance

Ramachandran Plots topical map of structure via and - see which angles are deemed
allowable
Repeating values of and along the chain result in regular structure
Van der Waals Radii, Bond angles functional group/side chain considerations
All play a role in understanding how
a AA chain of aa form a complex
structure

R groups face
outward from the
center pole, side
chains interact

Protein Secondary Structure

The residue by residue conformation of the peptide backbone in a protein
o a-helix (left or right-handed
o B-sheets (parallel and antiparallel)
a-helix:
o simplest structure that a polypeptide could retain
o R groups face outward from an imaginary longitudinal axis
o Single turn = ~5.4 A (A=0.1nm)
o aa resides that form a-helix have = - 60o and = -45o -50o
o 3.6 aa to each turn

o A-helix in proteins is right-handed

Assigning a left or right handed a-helix (rule of thumb)

What makes a-helix a stable conformation?

o Utilization of internal H-H bonding
o Via the H- of the N- towards that of the carboxy oxygen atom of the aa of toward the completion of one
turn
Occurs at every peptide bond
3-4 H bond in single turn
stability

Left handed a-helixes are rare in proteins

a-helix will only form with same stereo isomeric series: L-aa vs D-aa cannot be mixed
Sequence and amino acid stability
o aa R group interactions stabilize or destabilize structure
o Glu repetitive sequence destabilized a-helix -ve groups cause resides to repeal (internal H-H
bonding cannot compensate)
o As will Arg/Lys repetitive sequence repulsion of + ve charges

Stabilizing ionic pair that indicates AA forming

Pro and Gly constraint a-helix formation

o Pro rotation at N-Ca is not possible How does this effect formation and Why?
o Gly not common in a-helices, high level of conformational flexibility Tighter coiled structure then
normal
o Net dipole of a-helix with chain length can be destabilizing thus to compensate this 4-ve aa
commonly found at N-terminus while 4 +ve aa at C-terminal
Summary of constraints that affect a-helix stability
o Electrostatic repulsion/attraction via R charged groups
o Size of adjacent R groups
o Interaction b/w R groups 3-4 residue apart
o aa interactions at the ends of the a-helix
Thus fully dependent on aa sequence and chemical functionality
B-conformation organizes polypeptide chains into sheets
o Repetitive structure
o Extended conformation forms a zigzag, layer chains forms pleats
B-sheets
o H-H bonding formed between adjacent rows
o R groups of adjacent aa protrude in opposite directions
o alternating patternParallel B-sheets: same amino to carboxyl orientation

Anti-parallel b-sheets: different/opposite amino to carboxyl orientation

Repetitive values in the region of =-110 to -140 and = +110 to +135 give extended chains with
conformations that allow interactions between closely folded parallel segments (B-sheet structures)
The B-sheet is characterized by H-H bonds crossing between chains
On average parallel (both cains running N-terminal to C-terminal in the same direction) sheet chains
have , = -119,113
On average, antiparallel B-sheet chains have , = -139,135.

Circular Dichroism (CD) rotational properties in polarized light a-heliz and B-conformations have specific
CD characteristics

Majority of peptide bonds in trans configuration

6% of Pro, Most located in B turns
B-turns, acommon occurrence in protein structures
Protein structure: 1/3 aa = turns or loops
Connects a-helix and b-sheet conformations
b-turns most common: connect 2 adjacent antiparallel b-sheets
Commonly found near surface of protein
Turn requires 4 aa
180o turn via the oxygen of the carbonyl group interactive with the H-Na on a 4th distant aa
Central 2 aa do no participate
Gly and Pro often found at b-turns
Why? Gly small/flexible, Pro imino in cis configuration insertion assists in adopting flexible region
Secondary structures demonstrate to have common
Bond angles ( and ) and aa content

Type 1 more common,

Type 2 always has

Gly at reside 3 in turn

Given a sequence-we can actually

predict the structure

Crystal structure of
TM0919, one of the 76
CASP6 target proteins. This
protein, whose function is
hydroperoxide resistance,
was entered into the Protein
Data Bank on August 17,
2004, after all predictions
on the target were
collected. (b) Comparison of
a successful prediction (red)
for TM0919 with the crystal
structure.

Protein Tertiary and Quaternary Structures

1o structure: amino acid (aa) sequence
2o structure: residue by residue conformation of the backbone of a polymer
3o structure: 3-D conformation of a polymer in its native folded state
Quaternary structure: 3D structure of a multi-subunit protein; particularly the manner which the
subunits fit/interact

Structural classification at the quaternary level

Fibrous Proteins longer polypeptide chains, arranged in long strands or sheets
o Predominately B-sheets
Globular Proteins long polypeptide chains arranged in a compact and globular form, highly folded and
spherical in shape
Complex structures can contain both a-helix and B-sheets
o Physical structural properties of each provide selective biochemical features
Structure and function
o Fiberous proteins: a-keratin; collagen, silkfibroin
o Globular proteins: Myoglobin, lysozyme
a-Keratin:
Strong cross-linking between polypeptide chains multihelical ropes, stabilized by disulfide bonding
Flexible
Simple repeating units retainng to 2 Structure (right-handed a-helix)
Insoluble in H2O
High number of Hydrophobic aa within internal and surface of structure i.e. Ala, Val, Leu, Ile, Met, Phe

Examples of a-Keratin: Hair, wool, nails, claws, quills, horns (<18% crossed-linked) hooves, outer skin
layer
Commonly called intermediate filament (IF) proteins
Yet different aa composition % provides ability to determine source of hair sheeps wool v. human
hair: amino acid composition analysis (CSI/Forensics)

The Perm

New growth has original S-S bond formation, thus perm is outgrown

Collagen
Connective tissues- tendons, cartilage, bone (<30 diff. structural variants)
Structurally different from the std a-helicies: left handed 3aa/turn, coiled coil combination of 3 super
twisted polypeptide in an opposing twist right handed strength
Superhelical twists is right handed
High % of Gly, Ala, Pro Hyp (4-trans-hydroxyproline; Post transitional modification of Pro)
(uncommon aa)
sequence within collagen: repeating tripeptide Gly-X-Y [mostly Pro; Y=4 Hyp]
Pro and 4Hyp that permits sharp structural twists; closing packing

Skin Fibroin
Produced by insects/spiders
Predominantly B conformation high % Ala and Gly
Very close packing of sheets interlocking R side chain extensive H-H bonding, highly optimized
vdW interactions
Silk does not stretch as b-conformations is in extended form
Structurally it is flexible as not stabilized via S-S bonding (vs/ a-Keratins)
Silk highly ordered structure

Notice
differences in
physicalchemical

Structural Diversity compact vs. extended

Contain the same number
of aa - surface to vol.
ratios

Understanding the Complexity of Globular Protein Structure

Myoglobin (Mr 16,700)
153 aa
O2-binding protein
Prosthetic group heme group (iron protoporphyrin
70% in a