Proteins

Learning Objectives
At the end of this lecture, you
will be able to
Define proteins
Describe the biomedical
importance of proteins
Know composition of proteins and
that basic monomeric unit of
proteins is amino acid
 Biomedical importance
Proteins are the most
abundant biological
macromolecules occurring in
all cells and all parts of cells
Proteins also occur in great
variety; thousands of different
kinds may be found in a single
 Proteins are the molecular
instruments through which
genetic information is
expressed
Biochemical catalysts known as
enzymes are proteins
Immunoglobulins that serve as
the first line of defense against
bacteria and viruses and other
foreign agents are proteins
 Several hormones are
proteins
Structural proteins
provide mechanical
support
Contractile proteins help
in the movement of muscle
fibre and microvilli
 Some proteins present in the cell
membrane, cytoplasm and
nucleus act as receptors
Certain other proteins in the cell
membrane act as channels and
transporters
The transport proteins carry
out the function of transporting
specific substances across the
membrane or in the body fluids
 Storage proteins bind with
specific substances and store them

Under certain conditions, proteins

can be catabolized to provide
energy

Proteins help in the maintenance

of water and electrolyte balance in
the body by exerting osmotic
pressure
 Hence proteins are the most
versatile macromolecules in living
systems and serve crucial functions
in essentially all biological processes
They function as catalysts, they
transport and store other molecules
such as oxygen, they provide
mechanical support and immune
protection, they generate
movement, and they control growth
and differentiation
 Several key properties enable
proteins to participate in such a wide
range of functions
1.
Proteins are linear polymers built of
monomer units called amino acids
The function of a protein is directly
dependent on its three dimensional
structure
Remarkably, proteins spontaneously fold
up into three-dimensional structures that
are determined by the sequence of amino
acids in the protein polymer
2. Proteins contain a wide range of
functional groups
These functional groups include
alcohols, thiols, thioethers,
carboxylic acids, carboxamides,
and a variety of basic groups
When combined in various
sequences, this array of functional
groups accounts for the broad
spectrum of protein function
3. Proteins can interact with one
another and with other biological
macromolecules to form complex
assemblies
The proteins within these assemblies
can act synergistically to generate
capabilities not afforded by the
individual component proteins
These assemblies include
macromolecular machines that carry
out the accurate replication of DNA,
4. Some proteins are quite rigid, whereas
others display limited flexibility
Rigid units can function as structural
elements in the cytoskeleton (the internal
scaffolding within cells) or in connective
tissue
Parts of proteins with limited flexibility may
act as hinges, springs, and levers that are
crucial to protein function, to the assembly of
proteins with one another and with other
molecules into complex units, and to the
transmission of information within and
between cells
 Composition of proteins
All proteins, whether from the
most ancient lines of bacteria
or from the most complex
forms of life, are constructed
from the same set of 20 amino
acids, covalently linked in
characteristic linear sequence
 Each of these amino acids
has a side chain with
distinctive chemical
properties
This group of 20 precursor
molecules maybe regarded
as the alphabet in which the
language of protein structure
is written
 Proteins are found in a wide
range of sizes, from relatively
small peptides with just a few
amino acid residues to huge
polymers with molecular weights
in the millions
Cells can produce proteins with
strikingly different properties and
activities by joining the same 20
amino acids in many different
 From these building blocks different organisms
can make such widely diverse products as
enzymes,
hormones,
antibodies,
transporters,
muscle fibers,
the lens protein of the eye,
feathers,
spider webs,
rhinoceros horn,
milk proteins,
antibiotics,
mushroom poisons
 Proteins are polymers of
amino acids, with each amino
acid residue joined to its
neighbor by a specific type of
covalent bond termed the
peptide bond
The term "residue" reflects
the loss of the elements of
water when one amino acid is
 Twenty different amino
acids are commonly found
in proteins
All of the 20 common
amino acids are amino
acids
They have a carboxyl group
and an amino group
 They differ from each other in
their side chains, or R groups,
which vary in
structure,
size,
electric charge,
and which influence the
solubility of the amino acids
 Non standard amino
acids

The 20 amino acids that

constitute the monomer units
of proteins are the standard
amino acids as the genetic
code specifies only these 20
L--amino acids
 In addition to these 20 amino acids
there are many less common ones
i.e., the non standard amino
acids
(for which the genetic code is not specified)

Some proteins contain additional

amino acids that arise by
modification of an amino acid
already present in a peptide i.e.,
after the protein has been
synthesized
 Examples include
conversion of peptidyl proline and
lysine to 4-hydroxyproline and 5-
hydroxylysine
the conversion of peptidyl glutamate
to -carboxyglutamate
the methylation, formylation,
acetylation, prenylation, and
phosphorylation of certain
aminoacyl residues
 Among these uncommon amino
acids
4-hydroxyproline, a
derivative of proline
5-hydroxylysine, derived from
lysine
The former is found in plant cell
wall proteins, and both are found
in collagen
 Another important
uncommon amino acid is
-carboxyglutamate,
found in the blood clotting
protein prothrombin and in
certain other proteins that
bind Ca2+ as part of their
biological function
 Some 300 additional amino acids
have been found in cells but not
as constituents of proteins
Examples include
Ornithine and citrulline
(intermediates in urea cycle)
Taurine (found in bile acids)
-Aminolevulinic acid (intermediate
in haem synthesis)
 D- AMINO ACIDS
D-amino acids are also non standard
amino acids that occur naturally
and include
free D- serine, and D-aspartate in brain
tissue
D-alanine & D-glutamate in the cell
walls of gram positive bacteria
D-amino acids are also found in
some antibiotics
 Selenocysteine, the 21st L--Amino
Acid?
Selenocysteine is an L--amino acid found in a
handful of proteins, including certain
peroxidases and reductases where it
participates in the catalysis of electron transfer
reactions
Its synthesis is not a post translational
modification, however, but a modification to a
serine that occurs while the serine is bound to
a unique RNA
The hydroxyl group of serine is replaced by a
selenium atom
 The selenocysteine is then inserted
into a protein as it is being
synthesized
Since selenocysteine is inserted into
polypeptides during translation, it is
commonly referred to as the "21st
amino acid"
However, unlike the other 20
genetically encoded amino acids,
selenocysteine is not specified by a
simple three-letter codon
 Chiral centres and
isomerism
For all the common
amino acids except
glycine (the R
group is another
hydrogen atom),
the carbon is
bonded to four
different groups:
a carboxyl
group,

an amino group,

an R group,

a hydrogen
atom
 The -carbon atom is thus a
chiral center and thus amino
acids have two possible
stereoisomers

All molecules with a chiral

center are also optically
active-that is, they rotate
 Special nomenclature has
been developed to specify the
absolute configuration of the
four substituents of
asymmetric carbon atom
The absolute configurations of
simple sugars and amino
acids are specified by the D, L
system, based on the
 Thus the amino
group of L-alanine
occupies the same
position about the
chiral carbon as
does the hydroxyl
group of L-
glyceraldehyde
Since they are non-
superimposable
mirror images of each
other, the two forms
represent a class of
Two conventions are used to
identify the carbons in an amino
acid
The additional carbons in an R group
are commonly designated , , and
so forth, proceeding out from the
carbon
For most other organic molecules,
carbon atoms are simply numbered
from one end, giving highest priority
to the carbon with the substituent
 Within this latter
convention, the
carboxyl carbon of an
amino acid would be
C-1 and the carbon
would be C-2
In some cases, such
as amino acids with
heterocyclic R groups
(such as histidine),
the Greek lettering
system is ambiguous
and the numbering
 For branched
amino acid side
chains,
equivalent
carbons are
given numbers
after the Greek
letters

Acidic and basic properties of amino acids

The form of an amino acid that has both a
positive and a negative charge is called a
zwitterion
Amino acids in solution at neutral pH exist
predominantly as dipolar ions (zwitterions)
 In this form, the amino group is
protonated (NH3+) and the
carboxyl group is deprotonated(
COO-)
A zwitterion can act either as an
acid (proton donor) or as a base
(proton acceptor)
Substances having this dual nature
are amphoteric and are often
called ampholytes
 Acting as acid (proton donor)

H H

R C COO- R C
COO- + H+

NH3+ NH2

Zwitterion
 Acting as base (proton acceptor)

H H

R C COO- + H+ R C
COOH

NH3+ NH3+

Zwitterion
 The ionization state of an
amino acid varies with pH
At physiological pH, the
carboxyl group is
dissociated , forming the
negatively charged
carboxylate ion (-COO ) and
-

the amino group is

 A simple monoamino
monocarboxylic - amino acid,
such as alanine, is a diprotic acid
when fully protonatedit has
two groups, the-COOH group and
the - NH3+ group, that can yield
protons
Each acid has a characteristic
tendency to lose its proton in an
aqueous solution
 The tendency of any acid (HA)
to lose a proton and form its
conjugate base (A-) is defined
by the equilibrium constant
(Keq) for the reversible
reaction

HA A - + H+
 Equilibrium constants for
ionization reactions are
usually called ionization
constants or acid dissociation
constants, often designated
pKa

The stronger the tendency

to dissociate a proton, the
stronger is the acid and
 pKa is the measure of the tendency of a
group to give up (donate) a proton with
the tendency decreasing ten-fold as the
pKa increases by one unit
Each titratable group has a pKa that
is numerically equal to the pH at
which exactly one half of the
protons have been removed from
that group
The pKa for the most acidic group
(COOH) is pK1, whereas the pKa for the
next most acidic group (NH3+) is pK2
 In acid solution (e.g., pH 1),
the amino group is protonated (
NH3+) and the carboxyl group is
not dissociated(COOH)
As the pH is raised, the
carboxylic acid is the first group
to give up a proton, as its pKa is
near 2
The dipolar form persists until
Titration of an amino
acid
Dissociation of the
carboxyl group:
The titration curve of an
amino acid can be
analyzed
Consider alanine, for
example, which
contains both an -
carboxyl and an -
amino group
 As the pH of the
solution is raised,
the -COOH group
of form I can
dissociate by
donating a proton
to the medium
The release of a
proton results in
the formation of
the carboxylate
 This structure is
shown as form II,
which is the dipolar
form of the
molecule

This form, also

called a zwitterion,
is the isoelectric
form of alanine-that
is, it has an overall
charge of zero
 The dissociation constant of the
carboxyl group of an amino acid is
called pK1 rather than pKa because the
molecule contains a second titratable
group
Dissociation of the amino group:
The second titratable group of alanine
is the amino group
This is a much weaker acid than the
-COOH group and, therefore, has a
much bigger dissociation constant, pK 2
 Release of a
proton from
the
protonated
amino group
of form II
results in the
fully
deprotonated
form of
 A simple amino acid e.g.
glycine is a diprotic acid
when fully protonated

This means that it has two

groups, the COOH and the
NH3+ group that can yield
protons
 Figure shows the titration
curve of the diprotic form of
glycine
The two ionizable groups of
glycine, the carboxyl group
and the amino group, are
titrated with a strong base
The plot has two distinct
stages, corresponding to
 At very low pH, the predominant ionic
species of glycine is the fully
protonated form,
+
H3N-CH2-COOH
At the midpoint of any titration, a point
is reached where the pH is equal to
the pKa of the protonated group being
titrated
At the midpoint in the first stage of the
titration, in which the -COOH group of
glycine loses its proton, equimolar
concentrations of the protonated
 As the titration proceeds, another
important point is reached at pH
5.97
This is a point, at which removal of
the first proton is essentially
complete and removal of the second
has just begun
At this pH glycine is present largely
as the dipolar ion (zwitterion)
+H N-CH -COO-
3 2
 The second stage of the titration
corresponds to the removal of a
proton from the NH3+ group of glycine
The pH at the midpoint of this stage is
9.60, equal to the pKa for the NH3
group
The titration is essentially complete at
a pH of about 12, at which point the
predominant form of glycine is
H2N-CH2-COO-
 From the titration curve of
glycine we can derive several
important pieces of
information
First, it gives a quantitative
measure of the pKa, of each of
the two ionizing groups:
2.34 for the -COOH group
9.60 for the NH3+ group
 The second piece of information
is that this amino acid has two
regions of buffering power
One of these is the relatively flat
portion of the curve, extending
for approximately 1 pH unit on
either side of the first pKa, of
2.6, indicating that glycine is a
good buffer near this pH
 The other buffering zone
is centered around pH
9.60

Note that glycine is not a

good buffer at the pH of
intracellular fluid or
blood, about 7.4
 Another important piece of information
derived from the titration curve of an
amino acid is the relationship between
its net charge and the pH of the
solution
At pH 5.97, glycine is present
predominantly as its dipolar form, fully
ionized but with no net electic charge
The characteristic pH at which the net
electric charge is zero is called the
isoeleetric point or isoelectric pH,
designated pI
 For glycine, which has
no ionizable group in its
side chain, the pI is
simply the arithmetic
mean of the two pKa
values
pI = (pK1+pK2)
 The farther the pH of a glycine
solution is from its pI, the greater the
net electric charge of the population
of glycine molecules
At any pH below its pI, glycine has a
net positive charge and will move
toward the negative electrode
(cathode)
At any pH above its pI, glycine will
have a net negative charge and thus
will move towards the positive
electrode (anode)
 All amino acids with a single -amino
group, a single -carboxyl group, and
an R group that does not ionize have
titration curves resembling that of
glycine
These amino acids have very similar,
although not identical, pKa values:
pKa of the COOH group in the range of
1.8 to 2.4
pKa of the -NH3+ group in the range of
8.8 to 1 1
 Alanine has only two dissociable
hydrogens (one from carboxyl &
one from amino group)
Its pI can be calculated as follows:
pI = pK1 + pK2 2
pK1 = 2.3 (for carboxyl group)
pK2 = 9.1 (for amino group)
pI = 2.3 + 9.1 2 = 5.7
 Amino acids with an
ionizable R group have more
complex titration curves,
with three stages
corresponding to the three
possible ionization steps and
thus they have three pKa
values
The additional stage for the
 Each of the acidic and basic
amino acids contains an
ionizable group in its side chain
Thus, both free amino acids and
some amino acids combined in
peptide linkages can act as
buffers
A buffer is a solution that resists
change in pH following the
addition of an acid or base
 The isoelectric points reflect
the nature of the ionizing R
groups present
For example, glutamate has a
pI of 3.22, considerably lower
than that of glycine
This is due to the presence of
two carboxyl groups, with an
average of their pKa values of
 Histidine has got 3
dissociable hydrogens
one from carboxyl,
pK1=1.8,

one from imidazole group,

pK2=6.0
one from amino group,
 The pI of Histidine, with two
groups that are positively
charged when protonated, is
7.59 (the average of the pKa
values of the amino and
imidazole groups)
Only histidine has an R group
(pKa = 6.0) providing
significant buffering power near
the neutral pH usually found in
IMPORTANCE OF pI
At physiologic pH, all amino
acids have both a negatively
charged group (-COO-) & a
positively charged group (-
NH3 )
+

At physiologic pH, all amino

acids are dipolar ions
(zwitterions)
Amino aids can act both as an
 The buffering capacity of
amino acids & proteins is
maximum nearest to their
pI values
The net charge on an amino
acid or a protein molecule
will be negative at pH
above the pI & positive at
Abbreviations and symbols for
the commonly occurring amino
acids
Each amino acid name has an
associated three-letter abbreviation
and a one-letter symbol
The one-letter codes are
determined by the following rules:
1. Unique first letter:

If only one amino acid begins

with a particular letter, then
that letter is used as its
symbol, for example,

I = isoleucine
 2. Most commonly
occurring amino
acids have priority:
If more than one
amino acid begins
with a particular
letter, the most
common of these
amino acids receives
this letter as its
symbol
For example, glycine
is more common than
glutamate, so G =
glycine
Similar
sounding
names:
Some one-letter
symbols sound
like the amino
acid they
represent
For example, F
=
phenylalanine,
Letter close to initial
letter:
For the remaining amino
acids, a one-letter symbol
is assigned that is as
close in the alphabet as
possible to the initial of
the amino acid
Further, B is assigned to
Asx, signifying either
aspartic acid or
asparagine, Z is assigned
to Glx signifying either
glutamic acid or
glutamine and X is
assigned to an
unidentified amino acid
Classification of amino acids
Amino acids can be classified on the
basis of four different criteria:
Side chains
Polarity of the side chain
Functional groups
Nutritional importance
Metabolic fate
However, some amino acid side
chains fit into a number of different
classifications and are therefore
grouped differently in different
Polarity of Bonds and Partial Charges
Polar bonds are covalent bonds in which
the electron cloud is denser around one
atom (the atom with the greater
electronegativity) than the other
Oxygen is more electronegative than
carbon, and a carbonoxygen bond is
therefore polar, with the oxygen atom
carrying a partial negative charge and
the carbon atom carrying a partial
positive charge
 In nonpolar carboncarbon bonds
and carbonhydrogen bonds, the
two electrons in the covalent bond
are shared almost equally
Nitrogen, also carries a partial
negative charge relative to carbon,
and the carbonnitrogen bond is
polarized
Sulfur can carry a slight partial
negative charge
SOLUBILITY
Water is a dipolar molecule
in which the oxygen atom
carries a partial negative
charge and the hydrogen
atoms carry partial positive
charges
For molecules to be soluble
in water, they must
contain charged or polar
groups that can associate
with the partial positive
and negative charges of
water
 Thus, the solubility of organic
molecules in water is
determined by both
the proportion of polar to
nonpolar groups attached to
the carbonhydrogen skeleton
to their relative positions in
the molecule
 Polar groups or molecules are called
hydrophilic (water-loving), and nonpolar
groups or molecules are hydrophobic
(water-fearing)
The water molecules interacting with a
polar or ionic compound form a hydration
shell around the compound
 Compounds that have large nonpolar
regions are relatively water insoluble
They tend to cluster together in an
aqueous environment and form weak
associations through van der Waals
forces, often termed hydrophobic bonds
 Hydrophobic compounds are essentially
pushed together as the water molecules
maximize the number of energetically
favorable hydrogen bonds they can form
with each other in the water lattice

Thus, lipids form droplets or separate

layers in an aqueous environment
A. Nonpolar, Aliphatic R
Groups

These are hydrophobic

The amino acids in this

group have non polar side
chains
 They do not participate in
hydrogen bonds

They are oily or lipid like and

promote hydrophobic
interactions

These side chains cluster

together in the interior of the
protein
 This class
includes
Glycine
Alanine
Proline
Valine
Leucine
Isoleucine
Methionin
e
 Electrons are shared equally
between the carbon and
hydrogen atoms in these side
chains, so that they cannot
hydrogen bond with water

Within proteins, these amino

acid side chains will cluster
together to form hydrophobic
cores
 Glycine is the simplest
amino acid, and it really
does not fit well into any
classification because its
side chain is only a
hydrogen atom
Because of this, it causes
the least amount of steric
hindrance in a protein
Therefore glycine is often
found in bends or in the
tightly packed chains of
fibrous proteins
 Alanine and the
branched chain amino
acids (valine, leucine,
and isoleucine) have
bulky, nonpolar,
aliphatic(open chain
hydrocarbon) side chains
Methionine, one of the
two sulfur-containing
amino acids, has a
nonpolar thioether group
in its side chain
 The role of proline in
amino acid structure
differ from those of the
nonpolar amino acids
The amino acid proline
contains a ring involving
its carbon and its -
amino group, which are
part of the peptide
backbone
It is an imino acid
This rigid ring causes a
kink in the peptide
B. Aromatic Amino Acids
The aromatic amino acids have
been grouped together because
they all contain ring structures
with similar properties, but their
polarity differs a great deal
The aromatic ring is a six-
membered carbonhydrogen
ring with three conjugated
double bonds (the benzene
 The substituents on this ring
determine whether the amino
acid side chain engages in
polar or hydrophobic
interactions
This group includes
Phenylalanine
Tyrosine
 In the amino acid phenylalanine, the
ring contains no substituents, and the
electrons are shared equally between
the carbons in the ring, resulting in a
very nonpolar hydrophobic structure
in which the rings can stack on each
other

In tyrosine, a hydroxyl group on the

phenyl ring engages in hydrogen
bonds, and the side chain is therefore
more polar and more hydrophilic
 The more complex ring structure in
tryptophan is an indole ring with a
nitrogen that can engage in hydrogen
bonds
Tryptophan is therefore also more
polar than phenylalanine
Tyrosine and tryptophan are
significantly more polar than
phenylalanine, because of the
tyrosine hydroxyl group and the
nitrogen of the tryptophan indole
ring
C. Aliphatic, Polar, Uncharged R
Groups
The R groups of these amino acids
are more soluble in water, or more
hydrophilic, than those of the
nonpolar amino acids, because
they contain functional groups that
form hydrogen bonds with water
These Amino acids have zero net
charge at neutral pH
 This class includes
Serine &
threonine which
contain a polar
hydroxyl group
Similarly side
chains of
asparagine &
glutamine
contain a carbonyl
group & amide
group, both of
 These side chains are sites of
attachment for other compounds
The polar hydroxyl groups of serine
& threonine serve as a site of
attachment for phosphate groups
The amide group of asparagine &
the hydroxyl group of serine &
threonine serve as a site of
attachment for oligosaccharide
chains in glycoproteins
 Amino acids with side chains
that contain an amide group
(asparagine and glutamine) or
a hydroxyl group (serine and
threonine) can be classified as
aliphatic, polar, uncharged
amino acids
Asparagine and glutamine are
amides of the amino acids
 As a consequence of their
hydrophilicity, these amino acids
are frequently found on the
surface of water-soluble globular
proteins

Cysteine, which is sometimes

included in this class of amino
acids, has been separated into
the class of sulfur-containing
amino acids
D. Sulfur-Containing Amino Acids
Both cysteine and methionine contain
sulfur
The side chain of cysteine contains a
sulfhydryl group that has a pKa of
approximately 8.4 for dissociation of its
hydrogen, so cysteine is predominantly
undissociated and uncharged at the
physiologic pH of 7.4
The polarity of cysteine is contributed by
its sulfhydryl group, which is a weak acid
 Cysteine is readily oxidized to form a
covalently linked dimeric amino acid
called cystine, in which two cysteine
molecules or residues are joined by a
disulfide bond
 The disulfide-linked residues are
strongly hydrophobic (nonpolar)
Disulfide bonds play a special role in
the structures of many proteins by
forming covalent links between parts
of a polypeptide molecule or between
two different polypeptide chains
 Methionine,
although it contains
a sulfur group, is a
nonpolar amino acid
with a large bulky
side chain that is
hydrophobic

It does not contain a

sulfhydryl group
 Its important and
central role in
metabolism is
related to its
ability to transfer
the methyl group
attached to the
sulfur atom to
other
E. The Acidic and Basic Amino Acids
The most hydrophilic R groups are those
that are either positively or negatively
charged
The amino acids aspartate and
glutamate have carboxylic acid groups
that carry a negative charge at physiologic
pH
The basic amino acids histidine, lysine,
and arginine have side chains containing
nitrogen that can be protonated and
positively charged at physiologic and lower
pH values
The amino acids in which the R groups have
significant positive charge at pH 7.0 are:
Lysine, which has a second primary amino group at
the position on its aliphatic chain
Arginine, which has a positively charged
guanidinium group
Histidine, which has an aromatic imidazole
group
 The side chains of the two basic
amino acids, arginine and lysine,
have pKa values above 10, so that
the positively charged form always
predominates at physiologic pH
The side chain of histidine (pKa ~
6.0) dissociates near physiologic pH,
so only a portion of the histidine side
chains carry a positive charge
Histidine is therefore weakly basic
& the free amino acid is mainly
 The amino acid side chains might
have very different pKas than
those of the free amino acids if
they are involved in hydrogen or
ionic bonds with other amino acid
side chains
Therefore, when histidine is
incorporated into a protein, its
side chain can be either
positively charged or neutral
Negatively Charged
(Acidic) R Groups

At neutral pH, the side chains

of these amino acids are fully
ionized, containing a
negatively charged
carboxylate group (--COO-)
 Therefore these
amino acids are
negatively
charged at
phyisologic pH
The two amino
acids having R
groups with a net
negative charge
at pH 7.0 are
aspartate and
 Humans have no dietary requirement for
protein, per se, but, the protein in food does
provide essential amino acids
Ten of the twenty amino acids needed for the
synthesis of body proteins are essential-
that is, they cannot be synthesized in
humans at an adequate rate
Of these ten, eight are essential at all times,
whereas two (arginine and histidine) are
required only during periods of rapid tissue
growth, characteristic of childhood or
recovery from illness
 Amino acids can be classified as
glucogenic or ketogenic based on
what intermediates are produced
during their catabolism
A. Glucogenic amino acids
Amino acids whose catabolism yields
pyruvate are termed glucogenic
or glycogenic
These intermediates are substrates
for gluconeogenesis and,
B. Ketogenic amino acids
Amino acids whose catabolism
yields either acetoacetate or
its precursor, (acetyl CoA or
acetoacetyl CoA) are termed
ketogenic
Acetoacetate is one of the
ketone bodies which also include
3-hydroxybutyrate and acetone
 Leucine and lysine are the
only exclusively ketogenic
amino acids found in proteins
Their carbon skeletons are not
substrates for gluconeogenesis
and, therefore, cannot give rise
to the net formation of glucose
or glycogen in the liver, or
glycogen in the muscles
 We now turn to polymers of
amino acids, the peptides and
proteins

Biologically occurring
polypeptides range in size
from small to very large,
consisting of two or three to
thousands of linked amino
acid residues
 Two amino
acid
molecules
can be
covalently
joined
through an
amide
 This linkage is formed by
removal of the elements of
water from the -carboxyl
group of one amino acid and
the -amino group of another
 Two configurations are
possible for a planar peptide
bond

In the trans configuration, the

two -carbon atoms are on
opposite sides of the peptide
bond

In the cis con-, these groups

 Almost all peptide
bonds in proteins
are trans
This preference for
trans over cis can
be explained by the
fact that steric
clashes between
groups attached to
the -carbon atoms
hinder formation of
the cis form but do
 The
peptide C-
N bonds,
have a
partial
double-
bond
character,
and thus
 Three amino acids can be joined by
two peptide bonds to form a
tripeptide; similarly, four amino acids
can be linked to form tetrapeptides,
five to form pentapeptides, and so
forth
When a few amino acids are joined in
this fashion, the structure is called an
oligopeptide
When many amino acids are joined,
the product is called a polypeptide
 Proteins may have thousands of
amino acid residues
Although the terms "protein" and
"polypeptide" are sometimes
used interchangeably, molecules
referred to as polypeptides
generally have molecular weights
below 10,000, and those called
proteins have higher molecular
weights
 The figure below shows the
structure of a pentapeptide
An amino acid unit in a peptide is
often called a residue (the part left
over after losing a hydrogen atom
from its amino group and the
hydroxyl moiety from its carboxyl
group)
 In a peptide, the amino acid residue
at the end with a free -amino group
is the amino-terminal (or N-terminal)
residue
The residue at the other end, which
has a free carboxyl group, is the
carboxyl-terminal (C-terminal)
residue
 When an amino acid
sequence of a peptide,
polypeptide, or protein is
displayed the aminoterminal
end is placed on the left, the
carboxyl-terminal end on the
right

The sequence is read left to

 Peptides contain only one free
-amino group and one free
-carboxyl group, at opposite
ends of the chain
These groups ionize as they
do in free amino acids,
although the ionization
constants are different
because an oppositely
 The -amino and -
carboxyl groups of all
non terminal amino
acids are covalently
joined in the peptide
bonds, which do not
ionize and thus do not
contribute to the total
 However, the R groups of some
amino acids can ionize, and in a
peptide these contribute to the
over all acid-base properties of
the molecule
Like free amino acids, peptides
have characteristic titration
curves and a characteristic
isoelectric pH (pI) at which they
do not move in an electric field
 The pKa value for an
ionizable R group can change
somewhat when an amino
acid becomes a residue in a
peptide
The Ioss of charge in the -
carboxyl and -amino
groups, the interactions with
other peptide R groups, and
 No generalizations can be made
about the molecular weights of
biologically active peptides and
proteins in relation to their
functions
Even the smallest peptides can
have biologically important
effects, e.g. the commercially
synthesized dipeptide l-aspartyl-
l-phenylalanine methyl ester, the
 Many small peptides exert their effects at
very low concentrations.
For example,a number of vertebrate
hormones are small peptides.
oxytocin (nine amino acid residues), which is
secreted by the posterior pituitary and
stimulates uterine contractions
thyrotropin-releasing factor (three residues),
which is formed in the hypothalamus and
stimulates the release of another hormone,
thyrotropin, from the anterior pituitary gland
Some extremely toxic mushroom poisons,
such as amanitin, are also small peptides,
as are many antibiotics
 At the other extreme is titin, a
constituent of vertebrate muscle,
which has nearly 27,000 amino
acid residues and a molecular
weight of about 3,000,000
The vast majority of naturally
occurring proteins are much
smaller than this, containing
fewer than 2,000 amino acid
residues
 Some proteins consist of a single
polypeptide chain, but others, called
multisubunit proteins, have two or
more polypeptides associated
noncovalently
The individual polypeptide chains in a
multisubunit protein may be identical or
different
If at least two are identical the protein is
said to be oligomeric, and the identical
units (consisting of one or more
polypeptide chains) are referred to as
protomers
 Hemoglobin,
for example, has
four polypeptide
subunits all held
together by
noncovalent
interactions:
two identical
chains
two identical
 Each subunit is
paired in an
identical way with
a subunit within
the structure of this
multisubunit
protein, so that
hemoglobin can be
considered either a
tetramer of four
polypeptide
 A few proteins contain two or more
polypeptide chains linked covalently
For example, the two polypeptide
chains of insulin are linked by
disulfide bonds
In such cases, the individual
polypeptides are not considered
subunits but are commonly referred
to simply as chains
 The amino acid composition of
proteins is also highly variable
The 20 common amino acids
almost never occur in equal
amounts in a protein
Some amino acids may occur
only once or not at all in a
given type of protein; others
may occur in large numbers
 We can calculate the approximate
number of amino acid residues in a
simple protein containing no other
chemical constituents by dividing
its molecular weight by 110
Although the average molecular
weight of the 20 common amino
acids is about 138, the smaller
amino acids predominate in most
proteins
 If we take into account the
proportions in which the various
amino acids occur in an average
protein, the average molecular
weight of protein amino acids is
nearer to 128
Because a molecule of water
(MW 18) is removed to create
each peptide bond, the average
molecular weight of an amino
acid residue in a protein is about
Classification of Proteins
Different classifications of proteins
are based on their:
1.Shape and Size
2.Biological actions
3.Solubility and physical
properties
4.Quality
 i. Classification of Proteins on
Shape and Size Basis
This classification is based on their
Axial Ratio [The ratio of molecular
length to molecular width]
i. Fibrous Proteins: Axial Ratio > 10.
e.g. Keratins, collagens, fibrinogen,
fibrin and elastin etc
ii. Globular Proteins: Axial Ratio < 10
[usually not > 3 or 4] e.g. myoglobin,
hemoglobin, ribonuclease and most of
ii. Functional Classification of Proteins
1. Catalytic Proteins:
These specialized proteins are called enzymes
which catalyze the biochemical reactions
2. Protective Proteins
a) Immunoglobins (Igs)
. These freely circulating proteins protect the body
from invading bacteria or viruses by engulfing
them
. In acquired immunodeficiency syndrome (AIDS) Igs
synthesized have no immunity against antigens
b) Fibrinogen
. This forms fibrin clot and stops bleeding from
wounds
3.Regulatory Proteins:
. Hormones control biochemical
reactions catalyzed by enzymes
either activating or inactivating
them through modification of
their molecules
. Protein hormones are
growth hormone
Insulin
Glucagon
4. Structural Proteins:
These proteins form various body
structures e.g.
Collagen
Elastin
Keratin

5. Transport Proteins:
These proteins transport various
substances from one part of the body to
the other e.g.
Hemoglobin transport O2 from lungs to
tissues and CO2 from tissues to lungs
Transferrin transports iron
6. Contractile Proteins:
These proteins are involved in muscle
contraction and relaxation
Myosin of thick filaments
Actin of thin filaments of skeletal muscles
7. Respiratory Proteins:
Heme containing proteins are involved
in the function of respiration e.g.
Hemoglobin
Myoglobin
cytochromes
8. Digestive Proteins:
These proteins are digestive
enzymes which digest our food
materials such as carbohydrates,
proteins, lipids and include
Amylase
Pepsin
Lipases etc
9. Toxin Proteins:
These proteins are hydrolytic
enzymes found in the venom of
poisonous snakes, sting of bees and
insects and hydrolyze the
compounds forming the structure of
the cell membrane
The poisonous mushrooms also have
such toxins
10. Storage Proteins:
These proteins store some
specific elements or compounds
with them
This is because of the presence of
the many binding sites in them for
the particular element e.g.
ferritin stores iron
ceruloplasmin stores copper
 iii. Classification of Proteins on
Solubility and Physicals Properties Basis
The following classification is based
upon physiochemical properties of
proteins
A protein may belong to one of the
three types, i.e.
A. Simple Proteins
B. Compound or Conjugated Proteins
C. Derived Proteins
A. Simple Proteins:
On hydrolysis, these proteins yield
only amino acids or their derivatives
These consist of the following types
1. Albumins: Soluble in water and
dilute salt solution
Animal Sources: Egg albumin,
lactalbumin, serum albumin
Vegetable Sources: Legumelin of peas,
leucosine of wheat
2. Globulins:
Sparingly soluble in water but soluble in dilute salt
solution
Heat coagulable
Examples include:
lactoglbulins,
myosin in muscle,
ovoglobulin of egg yolk,
lactglobulin,
serum globulin
legumin
3. Globins
They unite with heme to form hemoglobin
Hemoglobins of different species differ only
with respect to globin, and the heme part is
the same in all cases
4. Prolamines or Gliadins:
Insoluble in water, soluble in 70-80% ethanol
These are rich in proline but deficient in
lysine
Examples: Gliadin of wheat,
Zein of maize
5. Histones:
Very strongly basic proteins as
they are rich in arginine
Soluble in water but insoluble in
dilute ammonia
Readily heat coagulable
Examples: nucleohistones,
chromosomal nucleoproteins,
6. Protamines:
Basic proteins
Soluble in water
Examples:
Salmine,
Sardinine,
sturine of fish sperms
7. Albuminoids/Sclero
Proteins:
Insoluble in all solvents and
include
i. Collagen:
i. Deficient in trp but rich in gly
ii. Present in bones, skin, cornea
and all body structure
i. Elastin:
i. Rich in nonpolar amino acid
ala, leu, val and pro
ii. Formed in excess during
pregnancy in uterine elastic
tissue
ii.Keratin:
i. Rich in cys
B. conjugated proteins
These proteins contain
permanently associated
chemical components in
addition to amino acids
The non-amino acid part of a
conjugated protein is usually
called its prosthetic group
 Conjugated proteins are
classified on the basis of the
chemical nature of their
prosthetic groups for example,
Lipoproteins contain lipids,
glycoproteins contain sugar
groups
metalloproteins contain a
specific metal
 Some proteins contain
more than one prosthetic
group

Usually the prosthetic

group plays an important
role in the protein's
biological function
C. Derived Proteins:
This class of proteins
includes substances which
are derived from simple and
conjugated proteins
These proteins are
subdivided into primary and
secondary derived proteins
1.Primary Derived Proteins:
. These are synonymous with
denatured proteins
. A protein is called a native
protein if its amino acid
composition and molecular
conformation are unchanged
from that found in natural
states
2. Secondary Derived Proteins:
These substances are
intermediates formed in the
progressive hydrolysis of protein
molecules
They are of different sizes and
different amino acid composition
and are roughly grouped into
proteoses, peptones and peptides
according to their molecular size
iv. Quality of Proteins
Proteins containing all 20 amino
acids that can be absorbed are
called Complete or Grade-I
proteins
Other proteins, deficient in some
essential amino acids are called
Incomplete or Grade-II
proteins
 The Structure of Proteins
Four levels of protein structure
are commonly defined
 A description of all covalent
bonds (mainly peptide bonds and
disulfide bonds) linking amino
acid residues in a polypeptide
chain is its primary structure
The most important element of
primary structure is the
sequence of amino acid
residues
 Secondary
structure refers
to the folding of
short (3- to 30-
residue),
contiguous
segments of a
polypeptide into
particularly stable
arrangements of
amino acid
residues giving
 Tertiary structure
describes the
assembly of
secondary structural
units into larger
functional units such
as the mature
polypeptide and its
component domains
Tertiary structure
hence describes all
aspects of the three-
 When a
protein has
two or
more
polypeptid
e subunits,
their
arrangeme
nt in space
is referred
to as
 The primary structure of a
protein determines how it folds up
into its unique three-dimensional
structure, and this in turn
determines the function of the
protein
The function of a protein therefore
depends on its amino acid sequence
Thousands of human genetic
diseases have been traced to the
production of defective proteins
 The defect can range from a single
change in the amino acid
sequence (as in sickle cell anemia)
to deletion of a larger portion of
the polypeptide chain (as in most
cases of Duchenne muscular
dystrophy)
Thus we know that if the primary
structure is altered, the function of
the protein may also be changed
 Finally, on comparing functionally
similar proteins from different
species, we find that these
proteins often have similar amino
acid sequences
An extreme case is ubiquitin, a
76-residue protein involved in
regulating the degradation of
other proteins
The amino acid sequence of
 We will examine how a sequence of
amino acids in a polypeptide chain is
translated into a discrete, three-
dimensional protein structure
We emphasize five themes
First, the three-dimensional
structure of a protein is determined
by its amino acid sequence
Second, the function of a protein
depends on its structure
Third, an isolated protein usually exists
in one or a small number of stable
structural forms
Fourth, the most important forces
stabilizing the specific structures
maintained by a given protein are
noncovalent interactions

Finally, amid the huge number of

unique protein structures, we can
recognize some common structural
patterns that help to organize our
understanding of protein architecture
 The spatial arrangement of
atoms in a protein is called its
conformation
The possible conformations of a
protein include any structural
state it can achieve without
breaking covalent bonds
Of the many conformations that
are theoretically possible in a
protein containing hundreds of
 The conformations existing
under a given set of
conditions are usually the
ones that are
thermodynamically the
most stable

Proteins in any of their

functional, folded
 The chemical interactions
that stabilize the native
conformation include
disulfide (covalent) bonds
and the weak (noncovalent)
interactions:
Hydrogen bonds
Hydrophobic
 Many proteins do not have
disulfide bonds

Disulfide bonds are found

primarily in secreted,
extracellular proteins (for
example, the hormone
insulin)
 For the intracellular proteins
of most organisms, weak
interactions are especially
important in the folding of
polypeptide chains into their
secondary and tertiary
structures
The association of multiple
polypeptides to form
 About 200 to 460 kJ/mol are
required to break a single
covalent bond, whereas weak
interactions can be disrupted by a
mere 4 to 30 kJ/mol

Individual covalent bonds, such as

disulfide bonds linking separate
parts of a single polypeptide
chain, are clearly much stronger
than individual weak interactions
 Yet, because they are so
numerous, it is the weak
interactions that predominate as
a stabilizing force in protein
structure

In general, the protein

conformation that is most stable
is the one with the maximum
number of weak interactions
 On carefully examining the
contribution of weak interactions to
protein stability, we find that
hydrophobic interactions generally
predominate
The interior of a protein is generally
a densely packed core of
hydrophobic amino acid side chains
It is also important that any polar or
charged groups in the protein interior
have suitable partners for hydrogen
 Salt bridges, especially those
that are partly or entirely
buried, can thus provide
significant stabilization to a
protein structure.
Ionic interactions also Limit
structural flexibility and confer a
uniqueness to protein structure
that nonspecific hydrophobic
interactions cannot provide
 Most of the structural patterns
outlined so far reflect two simple
rules:
(1) hydrophobic residues are
largely buried in the protein
interior, away from water
(2) the number of hydrogen
bonds and ionic interactions
within the protein is
maximum
 Protein structure is stabilized
by multiple weak
interactions
Hydrophobic interactions are
the major contributors to
stabilizing the globular form of
most soluble proteins
Hydrogen bonds and ionic
interactions are optimized in
 The nature of the covalent
bonds in the polypeptide
backbone places
constraints on structure
The peptide bond has a
partial double-bond
character that keeps the
entire six-atom
peptide
Requirements of the Three-
Dimensional Structure

The overall three-dimensional

structure of a protein must
meet certain requirements to
enable the protein to function
in the cell or extracellular
medium of the body
 The first requirement is the
creation of a binding site that
is specific for just one
molecule, or a group of
molecules with similar
structural properties

The specific binding sites of a

protein usually define its role
 The three-dimensional structure
also must exhibit the degrees of
flexibility and rigidity appropriate
to its function
Some rigidity is essential for the
creation of binding sites and for a
stable structure (i.e., a protein
that just flopped all over the
place could not accomplish
anything)
 However, flexibility and mobility
in structure enables the protein
to fold as it is synthesized, and to
adapt as it binds other proteins
and small molecules
The three-dimensional structure
must have an external surface
appropriate for its environment
(e.g., cytoplasmic proteins need
to keep polar amino acids on the
 In addition, the conformation
must also be stable, with little
tendency to undergo refolding
into a form that cannot fulfill
its function or that
precipitates in the cell
Finally, the protein must have
a structure that can be
degraded when it is damaged
Requirements of a Protein
Structure
Function
Binding specificity
Flexibility
Solubility or lipophilicity
Stability
Degradability
SECONDARY STRUCTURE
Secondary structure, the folding
of short (3- to 30-residue),
contiguous segments of
polypeptide into geometrically
ordered units
The term secondary structure
refers to any chosen segment of a
polypeptide chain and describes
the local spatial arrangement of its
Peptide Bonds Restrict Possible Secondary
Conformations
Free rotation is possible about only two of
the three covalent bonds of the
polypeptide backbone:
the -carbon (C) to the carbonyl carbon
(Co) bond,
the C to nitrogen bond
 There thus is no freedom of rotation
about the bond that connects the
carbonyl carbon and the nitrogen of
a peptide bond
Consequently, the O, C, N and H
atoms of a peptide bond are
coplanar
 The imposed semi-rigidity of
the peptide bond has
important consequences for
the manner in which peptides
and proteins fold to generate
higher orders of structure
 The angle
about the C
N bond is
termed the
phi () angle,
and that
about the Co
C bond
the psi ()
 Regions of ordered secondary
structure arise when a series of
aminoacyl residues adopt similar phi
and psi angles

Extended segments of polypeptide

(eg, loops) can possess a variety of
such angles
 There are a few types of secondary
structure that are particularly stable
and occur widely in proteins
The most prominent are the helix
and conformations
Another common type is the turn
Where a regular pattern is not
found, the secondary structure is
sometimes referred to as undefined
or as a random coil
 The Helix is a common protein
secondary structure
The simplest arrangement the
polypeptide chain can assume, given
its rigid peptide bonds (but free
rotation around its other, single
bonds), is a helical structure, called
the helix
Generally, about one fourth of all
amino acid residues in proteins are
found in helices, the exact fraction
 The polypeptide
backbone of an
helix is twisted
by an equal
amount about
each -carbon
with a phi angle
of approximately
57 degrees and
a psi angle of
approximately
47 degrees
 In this
structure the
polypeptide
backbone is
tightly wound
around an
imaginary axis
drawn
longitudinally
through the
 The R
groups of
each
aminoacy
l residue
in an
helix face
outward
 Proteins contain
only L-amino
acids, for which
a right-handed
helix is by far
the more stable,
and only right-
handed helices
are present in
proteins
 The repeating
unit is a single
turn of the
helix, and each
helical turn
includes 3.6
amino acid
residue and the
distance it rises
per turn (its
pitch) is 0.54
nm
 The structure is
stabilized by a
hydrogen bond
between the
hydrogen atom
attached to the
electronegative
nitrogen atom of a
peptide linkage and
the electronegative
carbonyl oxygen
atom of the fourth
 Within the
helix, every
peptide
bond
(except
those close
to each end
of the helix)
participates
in such
hydrogen
 Each successive turn of the
helix is held to adjacent turns by
three to four hydrogen bonds,
conferring significant stability on
the overall structure

The ability to form the maximum

number of hydrogen bonds,
supplemented by van der Waals
interactions in the core of this
 Many helices have
predominantly
hydrophobic R groups on
one side of the axis of the
helix and predominantly
hydrophilic ones on the
other
These amphipathic
helices are suitable for
the formation of
interfaces between polar
and nonpolar regions such
as the hydrophobic
 Not all polypeptides can form a
stable helix
Each amino acid residue in a
polypeptide has an intrinsic
propensity to form an a helix,
reflecting the properties of the R
group and how they affect the
capacity of the adjoining main-
chain atoms to take up the
characteristic angles
Alanine shows the greatest
 The position of an amino
acid residue relative to its
neighbors is also important

Interactions between amino

acid side chains can
stabilize or destabilize the
-helical structure
 For example, if a polypeptide
chain has a long block of
Glutamine residues, this
segment of the chain will not
form an helix at pH 7.0
The negatively charged
carboxyl groups of adjacent
GIutamine residues repel each
other so strongly that they
 For the same reason, if there are
many adjacent Lys and/or Arg
residues, with positively charged
R groups at pH 7.0, they also
repel each other and prevent
formation of the helix

The bulk and shape of Ser, Thr,

and Cys residues can also
destabilize an helix if they are
close together in the chain
 Another constraint on the
formation of the helix is the
presence of Proline or Glycine
residues
In proline, the nitrogen atom is
part of a rigid ring, and rotation
about the N-C bond is not possible
Thus, a proline residue introduces
a destabilizing kink in an helix
For this reason, proline is only
rarely found in an helix
 Glycine occurs
infrequently in helices for
a different reason
It has more conformational
flexibility than the other
amino acid residues
Polymers of glycine tend to
take up coiled structures
 In summary, four types of constraints
affect the stability of an helix:
(1) The intrinsic propensity of an amino acid
residue to form an helix
(2) The interactions between R groups,
particularly those spaced three (or four)
residues apart
( 3) The bulkiness of adjacent R groups
( 4) The occurrence of Pro and Gly residues

The tendency of a given segment of a

polypeptide chain to form an helix
therefore depends on the identity and
sequence of amino acid residues within
 The Conformation organizes
polypeptide Chains in sheets
Sheets are a second type of regular
secondary structure that maximizes
hydrogen bonding between the peptide
backbones while maintaining the
allowed torsion angles

The backbone of the polypeptide chain

is extended into a zigzag rather than
helical structure
 The zigzag polypeptide chains
can be arranged side by side
to form a structure resembling
a series of pleats

In this arrangement called a

sheet, hydrogen bonds form
between adjacent segments of
polypeptide chain
 The -pleated sheet is described
as parallel if the polypeptide
strands run in the same direction
(as defined by their amino and
carboxy terminals) and anti-
parallel if they run in opposite
direction

Antiparallel strands are often the

same polypeptide chain folded
 The amino acid side chains
of each polypeptide strand
alternate between extending
above and below the plane
of the -sheet

Most sheets are not perfectly

flat but tend to have a right-
handed twist
 The individual segments that
form a sheet are usually
nearby on the polypeptide chain,
but can also be quite distant from
each other in the linear sequence
of the polypeptide and may even
be in different polypeptide chains
Clusters of twisted strands of
sheet form the core of many
globular proteins
C. Nonrepetitive Secondary
Structures
-Helices and -pleated sheets are
patterns of regular structure with a
repeating element, the turn of a
helix or a pleat

In contrast, bends, loops, and turns

are nonregular secondary
structures that do not have a
repeating element
 Roughly half of the residues in a
"typical" globular protein reside in
helices and sheets and half in loops,
turns, bends, and other extended
conformational features

They are characterized by an abrupt

change of direction and are often found
on the protein surface

For example, -turns are short regions

usually involving four successive amino
acid residues
 Turns are common in Proteins
In globular proteins, which have a
compact folded structure, nearly
one-third of the amino acid
residues are in turns or loops where
the polypeptide chain reverses
direction

These are the connecting elements

that link successive runs of helix
or sheets
 The structure is a 180O turn
involving four amino acid
residues, with the carbonyl
oxygen of the first residue
forming a hydrogen bond with
the amino-group hydrogen of
the fourth
The peptide groups of the
central two residues do not
participate in any inter-
 Glycine and Proline residues
often occur in turns
Glycine because it is small
and flexible
Proline because peptide bonds
involving the imino nitrogen
readily assume the cis
configuration, a form that is
particularly amenable to a
 Loops or random coils are
irregular in conformation, and
serve key biologic roles

While loops lack apparent

structural regularity, they are
neither truly disordered
nor random
 They are stabilized through
specific hydrogen bonds
dictated by the primary
sequence of the protein

The nonregular coils, loops,

and other segments are usually
more flexible than the
relatively rigid helices and
-pleated sheets
Supersecondary structure
A motif also called a
supersecondary structure or
fold is simply a recognizable
folding pattern involving two or
more elements of secondary
structure and the connection(s)
between them

Structural motifs are

 A motif can be very simple, such
as two elements of secondary
structure folded against each
other, and represent only a small
part of a protein
An example is a -- loop
A motif can also be a very
elaborate structure involving
scores of protein segments folded
together, such as the barrel
The tertiary structure
The tertiary structure of a protein is
the folding pattern of the secondary
structural elements into a three-
dimensional conformation
It indicates, in three-dimensional
space, how secondary structural
featureshelices, sheets, bends,
turns, and loopsassemble to form
domains and how these domains
relate spatially to one another
 Domains in the Tertiary
Structure
The tertiary structure of large
complex proteins is often described
in terms of physically independent
regions called structural domains

You can usually identify domains

from visual examination of a three-
dimensional figure of a protein
 Each domain is formed from a
continuous sequence of amino
acids in the polypeptide chain that
are folded into a three-
dimensional structure
independently of the rest of the
protein

Two domains are connected

through a simpler structure like a
loop
 The structural features of each
domain can be discussed
independently of another domain in
the same protein, and the structural
features of one domain may not
match that of other domains in the
same protein
A domain is a section of protein
structure sufficient to perform a
particular chemical or physical task
such as binding of a substrate or
other ligand
Quaternary structure
Some proteins contain two
or more separate
polypeptide chains, or
subunits, which may be
identical or different
The arrangement of these
protein subunits in three-
dimensional complexes
 Many proteins have multiple
polypeptide subunits (from two to
hundreds)
The association of polypeptide chains
can serve a variety of functions
A multisubunit protein is also referred
to as a multimer, with the prefixes
homo or hetero used to describe
identical or different subunits,
respectively
A multimer with just a few subunits is
often called an oligomer
 If a multimer has nonidentical
subunits, the overall structure of
the protein can be asymmetric
and quite complicated

However, most multimers have

identical subunits or repeating
groups of nonidentical subunits,
usually in symmetric
arrangements
 Insulin is composed of two
nonidentical polypeptide chains
attached to each other through
disulfide bonds between the
chains

The subunits of globular proteins

are generally not held together
by disulfide bonds, but regions of
the same chain may be
 Insulin actually fits this generalization
because it is synthesized as a single
polypeptide chain, which forms the
disulfide bonds

Subsequently, a proteolytic enzyme in

secretory vesicles clips the polypeptide
chain into two nonidentical subunits

Generally, each subunit of most

protomers and oligomers is synthesized
as a separate polypeptide chain
 Protein Folding
Proteins are dynamic molecules that can
fold into their functionally competent
conformation in milliseconds, and
sometimes can refold if their conformation
becomes disrupted, or denatured
How is this remarkable process of folding
achieved?
Folding into the native state does not
involve a haphazard search of all possible
structures
Denatured proteins are not just random
coils
 The Native Conformation of a Protein
Is Thermodynamically Favored
The number of potential conformations of
even a relatively small15-kDa
polypeptide is unbelievably vast
Proteins are guided through this vast
labyrinth of possibilities by
thermodynamics
Since the biologically relevantor native
conformation of a protein generally is
that which is most energetically favored,
knowledge of the native conformation is
specified in the primary sequence
 However, if one were to wait
for a polypeptide to find its
native conformation by
random exploration of all
possible conformations, the
process would require billions
of years to complete
Clearly, protein folding in cells
takes place in a more orderly
and guided fashion
 Protein folding generally
occurs via a stepwise process
In the first stage, as the newly
synthesized polypeptide
emerges from the ribosome,
short segments fold into
secondary structural units that
provide local regions of
organized structure
 Then, each element of
secondary or super-secondary
structure facilitates proper
folding by directing the folding
process toward the native
conformation and away from
unproductive alternatives

For oligomeric proteins,

individual protomers tend to
 Auxiliary Proteins Assist
Folding
Under appropriate laboratory
conditions, many proteins will
spontaneously refold after being
denatured (i.e., unfolded) by
treatment with acid or base,
chemotropic agents, or detergents
However, refolding under these
conditions is slowminutes to
hours
 A loss of three-dimensional
structure sufficient to cause loss
of function is called denaturation
The denatured state does not
necessarily equate with complete
unfolding of the protein and
randomization of conformation
Under most conditions,
denatured proteins exist in a set
of partially folded state
 Certain proteins denatured by
heat, extremes of pH, or
denaturing reagents will regain
their native structure and their
biological activity if returned to
conditions in which the native
con-formation is stable

This process is called

renaturation
 Purified ribonuclease A denatures
completely in a concentrated urea
solution in the presence of a
reducing agent
Denaturation of ribonuclease is
accompanied by a complete loss of
catalytic activity
When the urea and the reducing
agent are removed, the randomly
coiled, denatured ribonuclease
spontaneously refolds into its
 Moreover, some proteins fail to
spontaneously refold in vitro,
often forming insoluble
aggregates, disordered
complexes of unfolded or
partially folded polypeptides
held together by hydrophobic
interactions

Cells employ auxiliary proteins

 Chaperone proteins participate in
the folding of over half of
mammalian proteins
The hsp70 (70-kDa heat shock
protein) family of chaperones binds
short sequences of hydrophobic
amino acids in newly synthesized
polypeptides
Chaperones prevent aggregation,
thus providing an opportunity for the
formation of appropriate secondary
structural elements and their
 The hsp60 family of chaperones,
sometimes called chaperonins, differ in
sequence and structure from hsp70 and
its homologs
Hsp60 acts later in the folding process,
often together with an hsp70 chaperone
The central cavity of the donut-shaped
hsp60 chaperone provides a sheltered
environment in which a polypeptide can
fold until all hydrophobic regions are
buried in its interior, eliminating
aggregation
 The transmissible spongiform
encephalopathies, or prion
diseases, are fatal neurodegenerative
diseases characterized by spongiform
changes, astrocytic gliomas, and
neuronal loss resulting from the
deposition of insoluble protein
aggregates in neural cells
They include CreutzfeldtJakob disease
in humans, scrapie in sheep, and
bovine spongiform encephalopathy
(mad cow disease) in cattle
 Prion diseases may manifest
themselves as infectious,
genetic, or sporadic
disorders
Because no viral or bacterial
gene encoding the
pathologic prion protein
could be identified, the
source and mechanism of
 Today it is recognized that
prion diseases are protein
conformation diseases
transmitted by altering the
conformation, and hence the
physical properties, of a
normal host protein
Human prion-related protein,
PrP, a glycoprotein encoded
on the short arm of
 The noninfectious form of PrP,
having the same amino acid and
gene sequences, is present on
the normal mammalian brain
No primary structure differences
or post translational
modifications have been found
between the normal and the
infectious forms of the protein
The key to becoming infectious
lies in changes in the three
 Pathologic prion proteins serve as
the templates for the conformational
transformation of normal PrP, known
as PrPc, into PrPsc
PrPsc is rich in sheet with many
hydrophobic aminoacyl side chains
exposed to solvent
As each new PrPsc molecule is
formed, it triggers the production of
yet more pathologic variants in a
conformational chain reaction
 Because PrPsc molecules associate
strongly with one other through
their exposed hydrophobic regions,
the accumulating PrPsc units
coalesce to form insoluble
protease-resistant aggregates
One pathologic prion or prion-
related protein can serve as
template for the conformational
transformation of many times its
number of PrPc molecules, prion
diseases can be transmitted by the
 Misfolding of proteins may occur
spontaneously or be caused by a
mutation in a particular gene, which
then produces an altered protein
In addition some apparently normal
proteins can, after abnormal
proteolytic cleavage, take on a
unique conformational state that
leads to the formation of long
fibrillar protein assemblies
consisting of pleated sheets
 Accumulation of these
spontaneously aggregating proteins
called Amyloids, has been
implicated in many degenerative
diseases, particularly in the
neurodegenerative disorder called
Alzeihmers Disease
The dominant component of the
Amyloid plaque that accumulates in
the Alzheimer's Disease is the
amyloid (A), a peptide
containing 40-42 amino acid
 This peptide, when aggregated in a
pleated sheet, is neurotoxic and
is responsible for the cognitive
impairment of the disease
The A that is deposited in the
brain in Alzheimer's Disease is
derived from the proteolytic
cleavage from the larger precursor
protein, a single transmembrane
protein expressed on the cell
surface on the brain
 The A peptides aggregate,
generating the amyloid that is
found in the brain parenchyma and
around blood vessels
A second factor involved in the
development of Alzheimer's disease
is the accumulation of
neurofibrillary tangles in the
brain
A key component of these tangled
fibers is an abnormal form of the
 In considering higher levels of
structure, it is useful to
classify proteins into two
major groups:
fibrous proteins, with
polypeptide chains arranged in
long strands or sheets
globular proteins, with
polypeptide chains folded into
a spherical or globular shape
 Fibrous proteins usually consist largely
of a single type of secondary
structure, and their tertiary structure
is relatively simple
Globular proteins often contain several
types of secondary structure
The two groups also differ functionally:
the structures that provide support,
shape, and external protection to
vertebrates are made of fibrous proteins
Whereas most enzymes and regulatory
proteins are globular proteins
 Fibrous proteins are
adapted for a Structural
function and possess axial
ratios (the ratio of their
shortest to longest
dimensions) of 10 or more
Fibrous proteins share
properties that give strength
and or flexibility to the
structures in which they occur
 All fibrous proteins are
insoluble in water, a
property conferred by a
high concentration of
hydrophobic amino acid
residues both in the
interior of the protein
and on its surface
 Tertiary Structure of Fibrous
Protein collagen, nicely
illustrates the relationship
between protein structure
and biological function

In this case, the

fundamental structural unit
is a simple repeating
 Collagen has evolved to
provide strength
It is found in connective
tissue such as tendons,
cartilage, the organic matrix
of bone, and the cornea of
the eye
Most abundant protein in
the body- 25% of the body
 The collagen super family of
proteins includes more than 20
collagen types

Collagens can be organized into

three groups based on their
location and functions in the
body
Fibril forming collagens
Type I, II, and III are the fibrillar
collagen
Type I are found in skin, bone,
tendons, blood vessels, and cornea
Type II are found in cartilages,
inter vertebral discs, and
vitreous body
Type III in blood vessels and
fetal skin
 Net work forming
collagens
They form a three dimensional
mesh, rather than distinct fibrils
Types IV and VII are included
in this group
Type IV present in basement
membranes
Type VII present beneath
epithelia
 Fibril associated
Type IX and XII bind to the
surface of collagen fibrils, linking
these fibrils to one another and
to other components in the
extracellular matrix
Type IX is present in cartilage
Type XII is present in tendons,
ligaments, and some other
tissues
 Although these molecules are
found throughout the body, their
types and organization are
dictated by the structural role
collagen plays in a particular organ

In some tissues, collagen may be

dispersed as a gel that give
support to the structure, as in the
extracellular matrix or the vitreous
humor of the eye
 In other tissues, collagen may be
bundled in tight, parallel fibers that
provide great strength, as in
tendons
In the cornea of the eye, collagen
is stacked so as to transmit light
with a minimum of scattering
Collagen of bone occurs as fibers
arranged at an angle to each other
so as to resist mechanical shear
from any direction
 A typical collagen molecule
is a long rigid right handed
triple helical structure
Each of the polypeptides in
the triple helix is known as
chain(different from -helix)
Each chain has three
amino acids per turn and is a
left handed helix
 The three polypeptide
chains are held together by
hydrogen bonds between
the chains
Variations in the amino acid
sequence of the chains
result in structural
components that are about
the same size (approx. 1000
 Structure of collagen
Collagen is rich in proline and
glycine
Glycine 35%
Alanine 11%
Proline and hydroxyproline 21%
The glycine residues are part of a
repeating sequence, -Gly-X-Y-,
X is frequently proline
Y is often hydroxyproline (but can be
hydroxylysine)
 Proline facilitates the formation
of the helical conformation of each
-chain because its ring structure
causes "kinks" in the peptide
chain
Glycine, the smallest amino acid,
is found in every third position of
the polypeptide chain
It fits into the restricted spaces
where the three chains of the helix
come together
 Collagen contains hydroxyproline
and hydroxylysine which are not
present in most other proteins
These residues result from
hydroxylation of some of the
proline and lysine residues as a
post translational
modification
Hydroxyproline is important in
stabilising the triple helical
structure by maximizing inter
 The hydroxyl group of the
hydroxylysine residues of
collagen may be
enzymatically glycosylated
Most commonly glucose
and galactose are
sequentially attached to the
polypeptide chain prior to
triple helix formation
Biosynthesis of collagen

The polypeptide precursors of the

collagen molecule are formed in
fibroblasts (or in the related
osteoblasts of bone and
chondroblasts of cartilage), and
are secreted into the extracellular
matrix

After enzymic modification, the

Formation of pro--chains:
Collagen is one of many proteins that
normally function outside of cells
Like most proteins produced for
export, the newly synthesized
polypeptide precursors of -
chains(pre-pro-collagen) contain a
special amino acid sequence at their
N-terminal ends
This acts as a signal that the
polypeptide being synthesized is
destined to leave the cell
 The signal sequence facilitates
the binding of ribosomes to the
rough endoplasmic reticulum
(RER), and directs the passage of
the polypeptide chain into the
cisternae of the RER

The signal sequence is rapidly

cleaved in the endoplasmic
reticulum to yield a precursor of
collagen called a pro-- chain
Hydroxylation:
The pro chains are processed by
a number of steps within the
lumen of the RER while the
polypeptides are still being
synthesized
Proline and lysine residues found in
the Y-position of the Gly-X-Y-
sequence can be hydroxylated to
form hydroxyproline and
hydroxylysine residues
 These hydroxylation
reactions require molecular
oxygen and the reducing
agent vitamin C (ascorbic
acid), without which the
hydroxylating enzymes,
prolyl hydroxylase and
hydroxylase, are unable to
 In the case of ascorbic acid
deficiency (therefore, a lack of prolyl
and lysyl hydroxylation), collagen fibers
cannot be cross-linked, greatly
decreasing the tensile strength of the
assembled fiber
One resulting deficiency disease is
known as scurvy
Patients with ascorbic acid deficiency
also often show bruises on the limbs as
a result of subcutaneous extravascation
of blood due to capillary fragility
Glycosylation:

Glucosyl and galactosyl

transferase attach glucosyl
or galactosyl residues to the
hydroxyl groups of specific
hydroxylysyl residues
Assembly and secretion:
After hydroxylation and glycosylation,
pro chains form procollagen, a
precursor of collagen that has a
central region of triple helix flanked
by the non-helical amino- and
carboxy- terminal extensions called
propeptides
The formation of procollagen begins
with formation of interchain disulfide
bonds between the C-terminal
extensions of the pro chains
 This brings the three -chains into
an alignment favorable for helix
formation
The procollagen molecules are
translocated to the golgi
apparatus, where they are
packaged in secretory vesicles
The vesicles fuse with the cell
membrane, causing the release of
procollagen molecules into the
extracellular space
 Extracellular cleavage of
procollagen molecules
After their release, the procollagen molecules are
cleaved by N- and C-pro-collagen peptidases,
which remove the terminal propeptides,
releasing triple-helical collagen molecules
 Formation of collagen fibrils:
Individual collagen molecules spontaneously
associate to form fibrils by forming an ordered,
overlapping, parallel array, with adjacent collagen
molecules, each overlapping its neighbor by a
length approximately three-quarters of a molecule
Cross-link formation:
The fibrillar array of collagen molecules
serves as a substrate for lysl oxidase
(copper is a co factor)
This extracellular enzyme oxidatively
deaminates some of the lysyl and
hydroxylysyl residues in collagen
The reactive aldehydes that result
(allysine and hydroxyallysine) can
condense with lysyl or hydroxylysyl
residues in neighboring collagen
molecules to form covalent cross-links
and thus mature collagen
 This cross-linking is essential
for achieving the tensile
strength necessary for the
proper functioning of
connective tissue
Therefore, any mutation that
interferes with the ability of
collagen to form cross-linked
fibrils almost certainly affects
the stability of the collagen
 Nutritional & Genetic
Disorders Can Impair
Collagen Maturation
The complex series of
events in collagen
maturation provide a model
that illustrates the biologic
consequences of incomplete
polypeptide maturation
 The best-known defect in collagen
biosynthesis is scurvy, a result of a
dietary deficiency of vitamin C
required by prolyl and lysyl
hydroxylases.
The resulting deficit in the number of
hydroxyproline and hydroxylysine
residues undermines the
conformational stability of collagen
fibers, leading to bleeding gums,
swelling joints, poor wound healing,
and ultimately death
 Menkes' syndrome,
characterized by kinky hair
and growth retardation,
reflects a dietary deficiency
of the copper required by
lysyl oxidase, which catalyzes
a key step in formation of the
covalent cross-links that
strengthen collagen fibers
 Ehlers-Danlos syndrome, a
group of connective tissue
disorders that involve impaired
integrity of supporting structures
that result in mobile joints and
skin

Defect is in the genes that encode

collagen
lysyl hydroxylase
 The most clinically important
mutations are found in the gene
for type III collagen

Collagen containing mutant

chains is not secreted, and is
either degraded or accumulated
to high levels in intracellular
compartments
 Because collagen type III is an
important component of the
arteries, potentially lethal vascular
problems occur

Although collagen type III is only a

minor component of the collagen
fibrils in the skin, for unknown
reasons, EDS patients also show
defects in collagen type I fibrils
Osteogenesis imperfecta (OI)
This disease, known as brittle bone
syndrome, is also a heterogeneous group of
inherited disorders distinguished by bones that
easily bend and fracture
Retarded wound healing and a rotated and
twisted spine leading to a "humped-back"
appearance are common features of the disease
Type I osteogenesis imperfecta is called
osteogenesis imperfecta tarda
This disease presents in early infancy with
fractures secondary to minor trauma, and may
be suspected if prenatal ultrasound detects
bowing or fractures of long bones
 Type II osteogenesis imperfecta
congenita, is more severe, and patients die in
utero or in the neonatal period of pulmonary
hypoplasia
Most patients with severe OI have mutations in
the gene for either the pro1- or pro 2- - chains
of type I collagen
The most common mutations cause the
substitution of single amino acids with bulky
side chains for the glycine residues that appear
as every third amino acid in the triple helix
The structurally abnormal pro chains can
prevent folding of the protein into a triple-
helical conformation
ELASTIN
In contrast to collagen, which forms fibers
that are tough and have high tensile
strength, elastin is a connective tissue
protein with rubber-like properties

They can be stretched to several times

their normal length, but recoil to their
original shape when the stretching force
is relaxed

Elastic fibers composed of elastin and

glycoprotein microfibrils are found in the
 Smaller quantities of elastin are
also found in skin, ear
cartilage, and several other
tissues

In contrast to collagen, there

appears to be only one genetic
type of elastin, although
variants arise by alternative
splicing
 Elastin is synthesized as a soluble
monomer of 70 kDa called
tropoelastin, which is a linear
polypeptide composed of about
700 amino acids that are
primarily small and nonpolar
(e.g., glycine, alanine, and valine)

Tropoelastin is secreted by the

cell into the extracellular space
 There it interacts with specific
glycoprotein microfibrils, such as
fibrillin, which function as a scaffold
onto which tropoelastin is deposited

Some of the prolines of

tropoelastin are hydroxylated to
hydroxyproline by prolyl
hydroxylase, though
hydroxylysine and glycosylated
hydroxylysine are not present
 Unlike collagen, tropoelastin
is not synthesized in a pro-
form with extension peptides

Furthermore, elastin does not

contain repeat Gly-X-Y
sequences, triple helical
structure, or carbohydrate
moieties
 After secretion from the cell, certain lysyl
residues of tropoelastin are oxidatively
deaminated to aldehydes by lysyl
oxidase, the same enzyme involved in
this process in collagen

However, the major cross-links formed in

elastin are the desmosines, which result
from the condensation of three of these
lysine-derived aldehydes with an
unmodified lysine to form a
tetrafunctional cross-link unique to elastin
 Once cross-linked in its mature,
extracellular form, elastin is highly
insoluble and extremely stable
and has a very low-turnover rate
Elastin exhibits a variety of
random coil conformations that
permit the protein to stretch and
subsequently recoil during the
performance of its physiologic
functions
 Deletions in the elastin gene
have been found in
approximately 90% of subjects
with the Williams- Beuren
syndrome, a developmental
disorder affecting connective
tissue and the central nervous
system
Fragmentation or, alternatively, a
decrease of elastin is found in
 Supravalvular aortic stenosis

SVAS results from an insufficiency of

elastin in the vessel wall, leading to
a narrowing of the large elastic
arteries

Mutations, by affecting synthesis of

elastin, probably play a causative
role in the supravalvular aortic
stenosis often found in this
condition
 Current theory suggests that
the levels of elastin in the
vessel walls may regulate the
number of smooth muscle cell
rings that encircle the vessel

If the levels of elastin are low,

smooth muscle hypertrophy
results, leading to a narrowing
and stenosis of the artery
Role of 1 antitrypsin in elastin
degradation
Blood and other body fluids contain a
protein, 1 -antitrypsin (currently also
called 1 antiprotinase) that inhibits a
number of proteolytic enzymes (also
called proteases or proteinases) that
hydrolyze and destroy proteins
The inhibitor was originally named 1-
antitrypsin because it inhibits the activity
of trypsin (a proteolytic enzyme
synthesized as trypsinogen by the
pancreas)
 1- antitrypsin comprises more
than ninety percent of the 1-
globulin fraction of normal plasma
1- antitrypsin has the important
physiologic role of inhibiting
neutrophil elastase - a powerful
protease that is released into the
extracellular space, and degrades
elastin of alveolar walls, as well as
other structural proteins in a
variety of tissues
 Most of the 1- antitrypsin found
in plasma is synthesized and
secreted by the liver

The remainder is synthesized by

several tissues, including
monocytes and alveolar
macrophages, which may be
important in the prevention of
local tissue injury by elastase
Role of 1- antitrypsin in the lungs:
In the normal lung, the alveoli are
chronically exposed to low levels of
neutrophil elastase released from
activated and degenerating neutrophils
This proteolytic activity can destroy the
elastin in alveolar walls if unopposed by
the inhibitory action of 1- antitrypsin, the
most important inhibitor of neutrophil
elastase
Because lung tissue cannot regenerate,
emphysema results from the destruction
of the connective tissue of alveolar walls
Emphysema resulting from 1- antitrypsin
deficiency:
In the United States, approximately two to five
percent of patients with emphysema are
predisposed to the disease by inherited defects
in 1- antitrypsin gene
A number of different mutations in the gene are
known to cause a deficiency of this protein, but
one single purine base mutation (resulting in
the substitution of lysine for glutamic acid at
position 342 of the protein) is clinically the most
widespread
An individual must inherit two abnormal alleles
to be at risk for the development of emphysema
 In a heterozygote, with one normal
and one defective gene, the levels of
1- antitrypsin are sufficient to protect
the alveoli from damage
A specific 1-AT methionine is required
for the binding of the inhibitor to its
target proteases
Smoking causes the oxidation and
subsequent inactivation of that
methionine residue, thereby
rendering the inhibitor powerless to
neutralize elastase
 Smokers with 1-AT deficiency,
therefore, have a considerably
elevated rate of lung destruction and
a poorer survival rate than non
smokers with the deficiency
The deficiency of elastase inhibitor
can be reversed by weekly
intravenous administration of 1-AT
The 1-AT diffuses from the blood into
the lung, where it reaches therapeutic
levels in the fluid surrounding the
lung epithelial cells
 FIBRILLINS ARE STRUCTURAL
COMPONENTS OF MICROFIBRILS

Microfibrils are fine fiber-like

strands 10 to 12 nm in diameter
which provide a scaffold for the
deposition of elastin in the ECM

Fibrillins are large glycoproteins

(about 350 kDa) that are major
structural component of these fibers
 They are secreted (subsequent to a
proteolytic cleavage) into the ECM by
fibroblasts and become incorporated
into the insoluble microfibrils

Fibrillin-1 is the main fibrillin

present, but fibrillins-2 and -3 have
also been identified, and fibrillin-2 is
thought to be important in deposition
of microfibrils early in development
 Other proteins including
microfibril-associated
proteins (MAGPs are also
associated with microfibrils

Fibrillin microfibrils are found in

elastic fibers and also in elastin-
free bundles in the eye, kidney,
and tendons
 Marfan Syndrome Is Caused by
Mutations in the Gene for Fibrillin-1

Marfan syndrome is a relatively

prevalent inherited disease affecting
connective tissue

It is inherited as an autosomal dominant

trait

Abraham Lincoln may have had this

condition
 It affects:

the eyes (eg, causing dislocation of the

lens, known as ectopia lentis),

the skeletal system (most patients are

tall and exhibit long digits
[arachnodactyly] and hyperextensibility
of the joints),

and the cardiovascular system (eg,

causing weakness of the aortic media,
leading to dilation of the ascending aorta)
 Most cases are caused by
mutations in the gene (on
chromosome 15) for fibrillin-1

This results in abnormal fibrillin

and/or lower amounts being
deposited in the ECM
 Mutations in the fibrillin-1 gene
have also been identified recently
as the cause of acromicric
dysplasia and geleophysic
dysplasia, which are characterized
by short stature, skin thickening,
and stiff joints

Congenital contractural
arachnodactyly is associated with
a mutation in the gene for fibrillin-2
Extra cellular matrix

Most mammalian cells are

located in tissues where they are
surrounded by a complex ECM
often referred to as connective
tissue, which protects the
organs and also provides
elasticity where required (eg, in
blood vessels, lungs, and skin)
 The ECM contains three major
classes of biomolecules:

1.Structural proteins, for example,

collagen, elastin, and fibrillin,

2. certain specialized proteins such

as fibronectin and laminin, which
form a mesh of fibers that are
embedded in

3.proteoglycans
 The ECM is not simply a glue
that holds cells together

It also serves to keep cells

from moving to other locations
and to prevent large molecules
and other particles, such as
microorganisms, from reaching
contiguous and distant cells
 This confining property of the
matrix is medically important
For example, infections spread, in
part, because the infectious agent
alters the containing capacity of
the ECM

Cancer cells that metastasize

(migrate to other tissues) can do so
only by altering the integrity of the
matrix
 Diseases such as rheumatoid
arthritis (an autoimmune
destruction of articular and
periarticular tissues) and
osteoarthritis (degenerative
joint disease often associated
with aging) involve damage to
the functional capacity of the
matrix
 Genetic defects may cause
components of the matrix to be
structurally and functionally
abnormal, resulting in connective
tissue disorders such as the

Ehlers- Danlos syndrome (caused

by a number of mutations that
affect specific collagen genes)

Marfans syndrome (a defect in the

protein, fibrillin)
 Alterations in the structural
characteristics of the matrix of the
renal glomerulus may allow proteins
to be excreted into the urine, an
indication of inexorable decline in
renal function

Deficiencies of lysosomal enzymes

involved in normal degradation of
molecules of the matrix result in
diseases such as the
mucopolysaccharidoses
 FIBRONECTIN IS AN
IMPORTANT GLYCOPROTEIN
INVOLVED IN CELL ADHESION
& MIGRATION

Fibronectin is a major
glycoprotein of the extracellular
matrix, also found in a soluble
form in plasma
 Fibronectin is also involved in cell
migration, as it provides a
binding site for cells and thus helps
them to steer their way through
the ECM

The amount of fibronectin around

many transformed cells is
sharply reduced, partly explaining
their faulty interaction with the
ECM
 LAMININ IS A MAJOR PROTEIN
COMPONENT OF BASAL LAMINAS

Basal laminas are specialized areas of

the ECM that surround epithelial and
some other cells (eg, muscle cells)

Laminin (a glycoprotein of about 850

kDa and 70 nm length) consists of three
distinct elongated polypeptide chains
(, , and chains) linked together to
form a complex, elongated shape
 In basal laminas, laminin forms
networks which are attached to type
IV collagen networks
The collagen interacts with laminin
(rather than directly with the cell
surface), which in turn interacts with
integrins thus anchoring the lamina
to the cells
 In the renal glomerulus, the basal
lamina consists of two separate sheets of
cells (one endothelial and one epithelial),
each disposed on opposite sides of the
lamina
These three layers make up the
glomerular membrane
This relatively thick basal lamina has an
important role in glomerular filtration,
regulating the passage of large
molecules (most plasma proteins) across
the glomerulus into the renal tubule
 The glomerular membrane allows
small molecules, such as inulin
(5.2 kDa), to pass through as
easily as water

On the other hand, only a small

amount of the protein albumin
(69 kDa), the major plasma
protein, passes through the
normal glomerulus
 This is explained by two sets of facts:
1. The pores in the glomerular
membrane are large enough to
allow molecules up to about 8 nm
to pass through
2. Albumin is smaller than this pore
size, but it is prevented from
passing through easily by the
negative charges of heparan
sulfate and of certain sialic acid-
containing glycoproteins present
in the lamina
 These negative charges repel albumin
and most plasma proteins, which are
negatively charged at the pH of blood

The normal structure of the

glomerulus may be severely damaged
in certain types of
glomerulonephritis (eg, caused by
antibodies directed against various
components of the glomerular
membrane)
 This alters the pores and the
amounts and dispositions of the
negatively charged
macromolecules referred to
above, and relatively massive
amounts of albumin (and of
certain other plasma proteins)
can pass through into the urine,
resulting in severe albuminuria
PROTEOGLYCANS &
GLYCOSAMINOGLYCANS

The Glycosaminoglycans
Found in Proteoglycans
Are Built Up of Repeating
Disaccharides

Proteoglycans are proteins

 The proteins bound covalently
to glycosaminoglycans are
called core proteins

The amount of carbohydrate

in a proteoglycan is usually
much greater than that found in
a glycoprotein and may
comprise up to 95% of its
 Figure show
the general
structure of
one
particular
proteoglyca
n,
aggrecan,
the major
type found
in cartilage
 It is very
large
(about 2
103 kDa),
with its
overall
structure
resembling
that of a
bottlebrush
 It contains a
long strand
of
hyaluronic
acid (one
type of GAG)
to which link
proteins are
attached
noncovale
ntly
 In turn, the
link proteins
interact
noncovalently
with core
protein
molecules
from which
chains of
other GAGs
(keratan
sulfate and
chondroitin
sulfate in this
case) project
 There are at least seven GAGs:
hyaluronic acid (hyaluronan),
chondroitin sulfate, keratan
sulfates I and II, heparin,
heparan sulfate, and
dermatan sulfate

GAGs are unbranched

polysaccharides made up of
repeating disaccharides, one
component of which is always an
 The other component of the
repeating disaccharide (except in the
case of keratan sulfate) is a uronic
acid, either l-glucuronic acid (GlcUA)
or its 5-epimer, l-iduronic acid (IdUA)

With the exception of hyaluronic acid,

all the GAGs contain sulfate groups,
either as O-esters or as N-sulfate (in
heparin and heparan sulfate)
 Hyaluronic acid is also
exceptional because it appears
to exist as a polysaccharide in
the ECM, with no covalent
attachment to protein, as the
definition of a proteoglycan
given above specifies
 Both GAGs and proteoglycans have
proved difficult to work with, partly
because of their complexity

However, since they are major

components of the ECM and have a
number of important biologic roles
as well as being involved in a
number of disease processes,
interest in them has increased
greatly in recent years
 Proteoglycans Are Important in
the Structural Organization of the
Extracellular Matrix

Proteoglycans are found in every

tissue of the body, mainly in the ECM
or ground substance

There they are associated with each

other and also with the other major
structural components of the matrix,
collagen and elastin, in specific ways
 Some proteoglycans bind to
collagen and others to elastin

These interactions are important

in determining the structural
organization of the matrix

Some proteoglycans (eg, decorin)

can also bind growth factors,
modulating their effects on cells
 In addition, some of them interact with
certain adhesive proteins such as
fibronectin and laminin , also found in the
matrix

The GAGs present in the proteoglycans

are polyanions and hence bind
polycations and cations such as Na+ and
K+

This latter ability attracts water by

osmotic pressure into the extracellular
matrix and contributes to its turgor
 GAGs also gel at relatively low
concentrations

Because of the long extended nature of the

polysaccharide chains of GAGs and their
ability to gel, the proteoglycans can act as
sieves, restricting the passage of large
nmacromolecules into the ECM but allowing
relatively free diffusion of small molecules

Again, because of their extended structures

and the huge macromolecular aggregates
they often form, they occupy a large
volume of the matrix relative to proteins
 Proteoglycans Are
Associated With Major
Diseases & With Aging

Hyaluronic acid may be

important in permitting tumor
cells to migrate through the
ECM
 Tumor cells can induce
fibroblasts to synthesize greatly
increased amounts of this GAG,
thereby perhaps facilitating
their own spread

Some tumor cells have less

heparan sulfate at their
surfaces, and this may play a
 The intima of the arterial wall contains
hyaluronic acid and chondroitin sulfate,
dermatan sulfate, and heparan sulfate
proteoglycans
Of these proteoglycans, dermatan sulfate
binds plasma low-density lipoproteins
In addition, dermatan sulfate appears to be
the major GAG synthesized by arterial
smooth muscle cells
Because it is these cells that proliferate in
atherosclerotic lesions in arteries,
dermatan sulfate may play an important role
in development of the atherosclerotic plaque
 In various types of arthritis,
proteoglycans may act as
autoantigens, thus contributing to
the pathologic features of these
conditions

The amount of chondroitin sulfate in

cartilage diminishes with age,
whereas the amounts of keratan
sulfate and hyaluronic acid increase
 These changes may contribute to
the development of osteoarthritis,
as may increased activity of the
enzyme aggrecanase, which acts to
degrade Aggrecan

Changes in the amounts of certain

GAGs in the skin are also observed
with aging and help to account for
the characteristic changes noted in
this organ in the elderly
 In the past few years it has
become clear that in addition to
their structural role in the ECM,
proteoglycans function as
signaling molecules which
influence cell behavior, and they
are now believed to play a part in
diverse diseases such as fibrosis,
cardiovascular disease and
cancer
Functions of GAGS and
proteoglycans
 In a globular protein, different
segments of the polypeptide
chain (or multiple polypeptide
chains) fold back on each other,
generating a more compact
shape than is seen in the fibrous
proteins
Globular proteins are compact,
roughly spherical molecules that
have axial ratios of not more
than 3
 The folding also provides the
structural diversity
necessary for proteins to
carry out a wide array of
biological functions
Globular proteins include
enzymes, transport proteins,
motor proteins, regulatory
proteins, immunoglobulins,
 Plasma Proteins
The concentration of total
protein in human plasma is
approximately
7.0-7.5g/dl
The proteins of the plasma are
actually a complex mixture that
includes not only simple
proteins but also conjugated
proteins such as glycoproteins
 The concentration of proteins in
plasma is important in
determining the distribution of
fluid between blood and tissues
The osmotic pressure (oncotic
pressure) exerted by the
plasma proteins is responsible
for attracting water back into
the circulation from the tissues
 In hypoprotinemia
fluid is not attracted
back into the
intravascular
compartment and
accumulates in the
extravascular tissue
spaces, a condition
 Because of the relative
ease with which they can
be obtained, plasma
proteins have been
studied extensively
One can separate plasma
proteins into three major
groups fibrinogen,
albumin, and globulins
 The most common method
of analyzing plasma
proteins is by
electrophoresis
It`s use permits resolution,
after staining, into five
bands, designated
albumin, 1, 2, and
 Most plasma proteins
are synthesized in the
liver
However the globulins
are synthesized in
plasma cells and certain
plasma proteins are
synthesized in other
 Most plasma proteins are
synthesized as preproteins
and contain amino terminal
signal peptides
They usually undergo
various posttranslational
modifications as they travel
through the cell
 Most plasma proteins are
glycoproteins
They generally contain
either N- or O- linked
oligosacharrides which have
different functions
Albumin is the major
exception and does not
contain sugar residues
 Each plasma protein has a
characteristic half life in
circulation

In certain diseases, half life

of a plasma protein may be
markedly altered
 The levels of certain plasma
proteins increase during
acute inflammatory states
or secondary to certain
types of tissue damage
These proteins are called
acute phase proteins and
include C- reactive
protein, 1-antitrypsin,
 The elevations of these
proteins vary from as little as
50% to as much as a 1000
fold in the case of CRP
Their levels are also usually
elevated during chronic
inflammatory states and in
patients with cancer
These proteins are believed to
 Functions of plasma
proteins
Antiproteases
Antichymotrypsin
1 Antitrypsin
Antithrombin
Blood clotting
Various coagulation factors
fibrinogen
 Enzymes
Functions in blood e.g., cholinestrase
Leakage from cells or tissues e.g.
aminotransferases
Hormones
Erythropoitin
Immune defense
Immunoglobulins
Complement proteins
2 microglobulins
 Inflammatory responses
Acute phase proteins e.g. CRP
Oncofetal
1 Fetoprotein
Transport or Binding proteins
Albumin binds various ligands e.g.
bilirubin, FFA, ions (Ca2+), metals (Cu2+ ,
Zn2+), methane, steroids, other
hormones and a variety of drugs
Cerruloplasmin binds Cu
Corticosteroid Binding Globulin
(transcortin) bind cortisol
 Haptoglobin binds extracorpuscular
haemoglobin
Lipoproteins (Chylomicrons, VLDL,
LDL, HDL) transport lipids
Hemopexin binds heme
Retinol Binding Protein binds Retinol
Sex Hormone Binding Globulin binds
testosterone, estradiol
Thyroid Binding Globulin binds T 4, T3
Transferrin transports iron
 Albumin
Albumin is the major protein of
human plasma and makes up
approximately 60% of the total
plasma protein
Its molecular weight is 69 kDa
and its concentration in plasma
is 3.4 to 4.7 g/dl
About 40% of albumin is present
in the plasma and the other 60%
in the extra cellular space
 The liver produces about
12g of albumin per day,
representing about 25% of
total hepatic protein
synthesis and half of its
secreted protein

Albumin is initially
synthesized as a
 Its signal peptide is
removed as it passes
through RER

A hexapeptide at the
resulting amino terminal is
subsequently cleaved
further along the secretory
pathway
 The mature human albumin
consists of one polypeptide
chain of 585 amino acids
and contains 17 disulfide
bonds

The synthesis of albumin is

depressed in a variety of
diseases, particularly those
 The plasma of patients with
liver disease often shows a
decreased albumin :
globulin ratio

The synthesis of albumin

decreases relatively early in
conditions of protein
 Because of it's relatively low
molecular weight and high
concentration, albumin is
responsible for 75-80% of
the osmotic pressure of the
human plasma

Electrophoretic studies have

shown that the plasma of
 Patients with this condition show
only moderate edema, probably by
an increase in the amounts of other
plasma proteins to compensate for
the lack of albumin

Another important function of

albumin is its ability to bind
various ligands, including free fatty
acids, calcium, certain steroid
hormones, bilirubin, drugs like
sulfonamides, penicillin G, dicumarol,
 Haptoglobin is a glycoprotein
that binds extracorpuscular
hemoglobin in a tight
noncovalent complex (Hb-Hp)
The amount of haptoglobin in
human plasma ranges from
40mg to 180mg of Hb binding
capacity
Approximately 10% of Hb that is
degraded each day is released
 The other 90% is present in old
damaged RBCs, which are degraded
by cells of the histiocytic system
The molecular mass of the Hb is
65kDa, and that of the Hp is
approximately 90kDa
Thus the combined mass of Hb-Hp
complex is 155kDa which is too large
to pass through the kidney glomeruli
Free Hb passes through the
glomerulus of the kidney, enters the
tubules, and precipitates
 The function of the Hp thus
appears to prevent loss of free
Hb into the kidney, which not
only protects the kidney
glomeruli but also conserves
valuable iron which would
otherwise be lost
The levels of Hp in human
plasma vary and are of some
diagnostic use
Low levels of Hp are found in
 This is explained by the fact that
whereas half life of Hp is 5 days,
the half life of Hp-Hb complex is
reduced to about 90 minutes, the
complex being rapidly removed
from the plasma by hepatocytes
Hp is an acute phase protein and
its plasma levels are elevated in
a variety of inflammatory states
 Transferrin (Tf) is a plasma
protein that plays a central role
in transporting iron around the
body to sites where it is needed
Tf is a 1 globulin with a
molecular mass of 76kDa
It is a glycoprotein and is
synthesized in the liver
About 20 polymorphic forms of Tf
have been identified
 It plays a central role in the
metabolism of iron because it
transports iron (2molecules of
Fe3+ per mole of Tf) in the
circulation to sites where it is
required
Approximately 200 billion RBCs
(about 20ml) are catabolized per
day, releasing about 25mg of iron
into the body - most of which will
be transported by Tf
 Cerruloplasmin (about 160
kDa)is an 2-globulin
It has a blue color because
of its high copper content
and carries 90% of the
copper present in plasma
Each molecule of
cerruloplasmin binds six
atoms of copper very tightly,
 Albumin carries the other 10%
of the plasma copper but
binds the metal less tightly
than does cerruloplasmin
Albumin thus donates its
copper to tissues more readily
than cerruloplasmin and
apears to be more important
than cerruloplasmin in copper
transport in the human body
 Cerruloplasmin exhibits
a copper dependant
oxidase activity, and is
involved in the
oxidation of Fe to Fe
2+ 3+

The amount of
cerruloplasmin in
plasma is decreased in
 In particular, low levels
are found in Wilson
Disease, a disease due
to abnormal metabolism
of copper in the human
body
Menkes Disease is
another condition
 1 Antiproteinase (about
52kDa) was formerly called
1 Antitrypsin
It is a single chain protein,
contains three
oligosacharride chains, and
is the major component
(>90%) of the 1 fraction of
the human plasma
 It is synthesized by
hepatocytes and macrophages
and is the principal serine
protease inhibitor of human
plasma

It inhibits trypsin, elastase and

certain other proteases by
forming complexes with them
 Most of the 1
antitrypsin is
synthesized by the liver,
but some is synthesized
by several tissues,
including monocytes
and alveolar
macrophages, which
 1 Antitrypsin has the
important physiological role of
inhibiting neutrophil
elastase a powerful protease
that is released into the
extracellular space, and
degrades elastin of alveolar
walls, as well as other
structural proteins in a variety
of tissues
 In the normal lung, the alveoli are
chronically exposed to low levels of
neutrophil elastase, released from
activated and degenerating neutrophils
This proteolytic activity can destroy the
elastin in alveolar walls if unopposed by
the inhibitory action of 1 antitrypsin
Lung tissue cannot regenerate and so
emphysema results from the
destruction of the connective tissue of
alveolar walls
 When the amount of 1
Antitrypsin is deficient and
polymorphonuclear white
blood cells increase in the
lung(e.g. during pneumonia),
the affected individuals lack
a check to proteolytic
damage of the lungs by
proteases such as elastase
 A particular methionine of
1 antitrypsin is involved in
its binding to proteases
Smoking oxidizes this
methionine and thus
inactivates it
As a result affected
molecules of 1 antitrypsin
 This is particularly
devastating in patients
who already have low
levels of 1 antitrypsin
Intravenous
administration of 1
antitrypsin has been
 The immunoglobulins
are synthesized mainly in
plasma cells, that are
specialized cells of B cell
lineage that synthesize
and secrete
immunoglobulins into the
plasma in response to
exposure to a variety of
 Immunoglobulins contain a
minimum of two identical light
(L)and two identical heavy (H)
chains, held together as a
tetramer (L2H2) by disulfide
bonds
The structure of IgG is Y shaped,
with antigen binding occurring at
both tips of the Y
Each chain can be divided
 The half of the light chain (L)
towards the carboxy terminal is
referred as the constant region
(CL), while the amino terminal
half is the variable region (VL)
of the light chain
Approximately one-quarter of the
heavy chain (H) at the amino
terminal is referred to as its
variable region (VH), and the
 The portion of the immunoglobulin
molecule that binds the antigen is
formed by the amino terminal
portions (variable regions) of both
the H and L chains, i.e. the VH and
VL domains
Digestion of an immunoglobulin by
the enzyme papain, produces two
antigen binding fragments (Fab)
and one crystallizable fragment
(Fc), which is responsible for
functions of immunoglobulins other
 Because there are two Fab regions,
IgG molecules bind two molecules of
antigen and are termed divalent
The site on the antigen to which an
antibody binds is termed an
antigenic determinant, or epitope
The area in which papain cleaves the
immunoglobulin molecule (the region
between the CH1and CH2 domains) is
referred to as the hinge region
 The hinge region confers flexibility
and allows both Fab arms to move
independently, thus helping them to
bind antigenic sites that may be
variable distance apart
The Fc and hinge regions differ in
different classes of antibodies but
overall model of antibody structure is
the same
There are two general types of light
chains, kappa () and lambda (),
which can be distinguished on the
 A given immunoglobulin
molecule always contains two
and two chains, never a
mixture of and

In humans chains are more

frequent than chains in
immunoglobulins

Five classes of H chains have

 They are designated , , ,
, and

The type of H chain thus

determines the class
immunoglobulins

There are thus five

immunoglobulin classes: IgG,
 Some immunoglobulins
such as IgG exist only
in the basic monomeric
structure, while others
such as IgA and IgM
can exist in higher
order polymers of two,
three (IgA), or five (IgM)
 The major function of IgG
immunoglobulins are:

Main antibody in secondary

response
Opsonizes bacteria making them
easier to phagocytose
Fixes complement which enhances
bacterial killing
Neutralizes bacterial toxins and
viruses
 IgA :
The only secreted antibody,
prevents attachment of
bacteria and viruses to mucous
membranes

IgM :
Produced in the primary
response to an antigen,
and does not cross the placenta
 IgD :
Functions are uncertain

IgE :
Mediates immediate
hypersensitivity by causing
release of mediators from mast
cells upon exposure to antigen
(allergan)
Defends against worm
 Both over and under production of
immunoglobulins may result in disease
states

Multiple myeloma is a neoplastic

condition in which there is either a large
increase of one particular
immunoglobulin or one particular light
chain (the latter termed a Bence jones
protein)

Decreased production may be restricted

to a single class of immunoglobulin
 A severe reduction in
synthesis of an
immunoglobulin class can
result in a serious
immunodeficency disease
e.g. aggamagloblinemia, in
which production of IgG is
markedly affected
 Proteins are subject to physical and
functional changes that mirror the life cycle
of the organisms in which they reside
A typical protein is:
born at translation,
matures through posttranslational processing
events such as partial proteolysis,
alternates between working and resting states
through the intervention of regulatory factors ,
ages through oxidation, deamidation, etc ,
and dies when it is degraded to its component
amino acids
 The life cycle of a hypothetical protein
(1) The life
cycle begins
with the
synthesis on
a ribosome
of a
polypeptide
chain,
whose
primary
structure is
dictated by
 (2) As
synthesis
proceeds
, the
polypepti
de
begins to
fold into
its native
conforma
tion
(blue)
 (3) Folding
may be
accompanied
by processing
events such
as proteolytic
cleavage of
an N
-terminal
leader
sequence
(Met-Asp-
Phe-Gln-Val))
or the
formation of
disulfide
bonds (SS)
 (4)
Subsequent
covalent
modificatio
n may, for
example,
attach a
fatty acid
molecule
(yellow) for
(5)
translocatio
n of the
modified
protein to a
 (6)
Binding an
allosteric
effector
(red) may
trigger the
adoption
of a
catalyticall
y active
conformati
on
 (7) Over
time,
proteins
become
damaged by
chemical
attack,
deamidation,
or
denaturation,
and (8) may
be "labeled"
by the
covalent
 (9) The
ubiquitinate
d protein is
subsequentl
y degraded
to its
component
amino acids,
which
become
available for
the
synthesis of
new proteins