Module-6-Proteins.pdf

Module-6-Proteins
Amino acids
Introduction
• There are approximately 300 amino acids present in
various animals, plants, and microbial systems, but only
20 amino acids are coded by DNA to appear in proteins.
• Cells produce proteins with strikingly different properties
and activities by joining the same 20 amino acids in many
different combinations and sequences.
• This indicates that the properties of proteins are
determined by the physical and chemical properties of
their monomer units, the amino acids.
Dr. Shiny C Thomas

Definition:
• Amino acids are the basic structural units of proteins
consisting of an amino group, (-NH2) a carboxyl (-COOH)
group a hydrogen (H) atom and a (variable) distinctive
(R) group.
• All of the substituents in amino acid are attached
(bonded) to a central α carbon atom. This carbon atom
is called α because it is bonded to the carboxyl (acidic)
group.
The general formula for the naturally occurring amino acids
would be :
H
|α Fig 5.1 : General formula of Amino acids
R –C –COOH
|
NH2

• A basic amino group (-NH2)
• An acidic carboxyl group ( -COOH)
• A hydrogen atom (-H)
• A distinctive side chain (-R)
In neutral solution (PH = 7), both the α- amino and α
carboxyl group are ionized resulting the
charged form of an amino acids called zwitterion (dipolar).
In dipolar (zwitterion) form the
amino group is protonated (-NH3
+) and the carboxyl group is
dissociated (deprotonated) (-COO-)
leading to a net charge zero.

Stereochemistry (Optical activity)
Stereochemistry mainly emphasizes the configuration of
amino acids at the α carbon atom, having either D or L-
isomers.
COOH COOH
| |
H - C – NH2 H2N – C – H
| |
R R
D (+) amino acid L (-) amino acid
Now because of its chirality it can have an L -form or a D –
form. Usually L -amino acids are incorporated into proteins.

The R group
can differ in its
size, it can
differ in its
shape, it can
differ in its
polarity.
There are twenty common amino acids which have
distinctive R groups with distinct properties of size, shape
and its polarity.

Classification of Amino Acids
L-Amino acids are the building blocks of proteins. They are
frequently grouped according to the chemical nature of their
side chains.
• Common groupings of amino acids are aliphatic,
hydroxyl/sulfur, cyclic, aromatic, basic, acidic and acid
amides. Links to individual amino acids are given below:
I. Structural Classification
This classification is based on the side chain radicals (R-
groups) as shown in the table 5.1.Each amino acid is
designated by three letter abbreviation eg. Aspartate as Asp
and by one letter symbol D.

• Acid-base or charge properties of amino acids: Amino
acids act as acids and bases. So they are called as
ampholytes or amphoteric substances
• amino acids have two ionizable groups (–COOH, NH3
+). The –COOH is several times more easily dissociates than
–NH3+.
• At neutral pH both groups are ionized, i.e., the
carboxyl group exist in dissociated form where as amino
group exist as associated form.
•
• This doubly charged molecule of amino acid containing
positive and negative charges is called as zwitter ion.

The charge of an amino acid always depends on the pH of its surroundings. In other words,
the charge of amino acid is altered by changing pH of its surroundings. This property is
exploited for the separation of amino acids. In strong acidic conditions (pH < 2) the –COOH
remains undissociated. When the pH is raised at pH of about 3 the proton from the
–COOH is lost –COO– is generated. This is called pK of acid group because at this pH
dissociated (–COO–) and undissociated (–COOH) species are found in equal amounts.
Similarly, if the pH is increased to 10, the amino group (–NH3
+) dissociates to –NH2 group.
This pH is called the pK of amino group of amino acid because at this pH associated
(–NH3
+) and dissociated (–NH2) species are present in equal amounts. (Fig. 2.11)
Therefore, an amino acid has two pK values corresponding to the two ionizable groups.
pK values indicates strength of each group. Further an amino acid exist as zwitter ion
at neutral pH and as cation at acidic pH and as anion at basic pH.

Example: For alanine, pKa is 2.4 and pKam is 9.7 (K is dissociation constant), the low pK
value of –COOH indicates more ionizing power.
Isoelectric pH: It is the pH at which the net charge of an amino acid is zero or when
the number of positive charges are equal to number of negative charges. At isoelectric
pH amino acids have minimum solubility. The isoelectric pH of an amino acid having
one amino group and one carboxyl group is equal to the arithamatic mean of pKa and
pKam values.

For most amino acids pI is close to 6.0. The situation differs for amino acids having
more than two ionizable groups. For example, glutamate is dicarboxylic acid so it can
have three pK values (two for carboxyl groups and one for amino group). Similarly, the
basic amino acid lysine can have three pK values (two for amino groups and one for carboxyl
group). In these cases, a different formula is used to obtain isoelectric pH. For
acidic amino acid like glutamate the isoelectric pH is equal to the half of sum of two
pK values of acidic groups.
For basic amino acid like lysine the isoelectric pH is equal to the half of sum of two
pK values of amino groups.

PEPTIDES
1. Peptides consist of 2 or more amino acid residues linked
by peptide bond.
2. A peptide bond is formed when carboxyl group of an
amino acid react with α-amino group of another amino acid.
(Fig. 2.12). Peptide bond formation between two amino
acids is always accompanied by loss of one water molecule.
Further, peptide and proteins contain an amino (N–)
terminus and carboxy (C–) terminus.
3. A peptide or protein is named starting with N-terminal
amino acid and usually the N-terminal is located on the left
hand side.

PROTEINS
• Proteins are present in every cell of humans, animals,
plant tissues, tissue fluids and in micro organisms.
• They account for about 50% of the dry weight of a cell. The
term protein is derived from the Greek word proteios
meaning holding first place or rank in living matter.
CHEMICAL NATURE OF PROTEINS
• All proteins are polymers of amino acids. The amino acids
in proteins are united through “Peptide” linkage.
• Sometimes proteins are also called as polypeptides
because they contain many peptide bonds.

CLASSIFICATION OF PROTEINS
There is no single universally satisfactory system of protein
classification so far.
1. One system classifies proteins according to their
composition or structure.
2. One system classifies them according to solubility.
3. One system classifies them according to their shape.
4. Classification of proteins based on their function also
found in literature.

Classification of proteins based on their composition
Proteins are divided into three major classes according to
their structure.
1. Simple proteins: Simple proteins are made up of amino
acids only. On hydrolysis, they yield only amino acids.
Examples: Human plasma albumin, Trypsin, Chymotrypsin,
pepsin, insulin, soyabean trypsin inhibitor and ribonuclease.
2. Conjugated proteins: They are proteins containing non-
protein part attached to the protein part.
The non-protein part is linked to protein through covalent
bond, non-covalent bond and hydrophobic interaction.
The non-protein part is loosely called as prosthetic group.

• On hydrolysis, these proteins yield non-protein
compounds and amino acids.
• Conjugated protein → Protein + Prosthetic group
• Eg: The conjugated proteins are further classified into
subclasses based on prosthetic groups.

3. Derived proteins: As the name implies this class of
proteins are formed from simple and conjugated proteins.
There are two classes of derived proteins.
(i) Primary derived proteins: They are formed from natural
proteins by the action of heat or alcohol etc. The peptide
bonds are not hydrolysed. They are synonymous with
denatured proteins.
Example: Coagulated proteins like cooked-egg albumin.
(ii) Secondary derived proteins: They are formed from
partial hydrolysis of proteins.
Examples: Proteoses, peptone, gelatin, and peptides.

Proteins classification according to their solubility
1. Albumins: Soluble in water and salt solutions.
Examples: Albumin of plasma, egg albumin and lactalbumin
of milk.
2. Globulins: Sparingly soluble in water but soluble in salt
solutions.
Examples: Globulins of plasma, ovoglobulins of egg,
lactoglobulin of milk.
3. Glutelins: Soluble in dilute acids and alkalies.
Examples: Glutenin of wheat, oryzenin of rice, zein of maize.
4. Protamins: Soluble in ammonia and water.
Examples: Salmine from salmon fish, sturine of sturgeon.

5. Histones: Soluble in water and dilute acids.
Example: Histones present in chromatin.
6. Prolamines: Soluble in dilute alcohol and insoluble in water
and alcohol.
Examples: Gliadin of wheat, zein of corn.
7. Sclero proteins: Insoluble in water and dilute acids and
alkalies.
Examples: Collagen, elastin and keratin.

Classification of proteins based on shape
Proteins are divided into two classes based on their shape.
1. Globular proteins: Polypeptide chain(s) of these
proteins are folded into compact globular (Spherical)
shape.
Examples: Haemoglobin, myoglobin, albumin, lysozyme,
chymotrypsin.
2. Fibrous proteins: Poly peptide chains are extended
along one axis.
Examples: α-keratin, β-keratin, collagen and elastin.

PROTEIN STRUCTURE
• Since proteins are built from amino acids by linking them
in linear fashion, it may be viewed as proteins having long
chain like structures.
• However, such arrangement is unstable and polypeptide
or protein folds to specific shape known as conformation,
which is more stable.
• Various stages involved in the formation of final
conformation from linear chain are divided into four
levels or orders of protein structure. They are:

1. Primary Structure
• The linear sequence of amino acid residues in a
polypeptide chain is called as primary structure.
• Generally disulfide bonds if any are also included in the
primary structure.
• Bonds responsible for the maintenance of primary
structure are mainly peptide bonds and disulfide bonds.
Both of them are covalent bonds

• 2. Secondary Structure
• Folding of polypeptide chain along its long axis is called as
secondary structure of protein.
• Folding of polypeptide chain can be ordered, disordered.
Secondary structure is often referred as conformation.
• So, proteins has ordered secondary structure or
conformation and random or disordered secondary
structure or conformation.

Ordered Conformation of Polypeptides
The polypeptide chain of some proteins may exist in highly
ordered conformation. The conformation is maintained by
hydrogen bonds formed between peptide residues.
Hydrogen bond
It is a weak ionic interaction between positively charged
hydrogen atom and negatively charged atoms like oxygen,
nitrogen, sulfur etc. It is indicated with broken lines (---).

There are two types of ordered secondary structure
observed in proteins.
1. The polypeptide chain of α-keratin, which is present in
hair, nails, epidermis of the skin is arranged as α-Helix. α-
letter is given to this type of structure because it was first
ordered structure noticed in proteins.
2. Polypeptide chain of β-keratin, which is present in silk
fibroin and spider web is arranged in β-pleated sheet. The β-
letter is given because it was observed later.

Secondary structure
• The next level of protein structure, secondary structure,
refers to local folded structures that form within a
polypeptide due to interactions between atoms of the
backbone. (The backbone just refers to the polypeptide
chain apart from the R groups – so all we mean here is
that secondary structure does not involve R group
atoms.)
• The most common types of secondary structures are the
α helix and the β pleated sheet. Both structures are held
in shape by hydrogen bonds, which form between the
carbonyl O of one amino acid and the amino H of another.

• In an α helix, the carbonyl (C=O) of one amino acid is
hydrogen bonded to the amino H (N-H) of an amino acid
that is four down the chain.
(E.g., the carbonyl of amino acid 1 would form a hydrogen
bond to the N-H of amino acid 5.)
• This pattern of bonding pulls the polypeptide chain
into a helical structure that resembles a curled ribbon, with
each turn of the helix containing 3.6 amino acids.
• The R groups of the amino acids stick outward from the
α helix, where they are free to interact .

Main Features of α-Helix
1. In α-helix polypeptide, backbone is tightly wound round
(coiled) long axis of the molecule.
2. The distance between two amino acid residues is 1.5 Å.
3. α-helix contain 3.6 amino acid residues per turn. The R-
group of amino acids project outwards of the helix (Fig).
4. The pitch of the α-helix is 5.4 Å long and width is 5.0 Å
(Fig).
5. The α-helix is stabilized by intra chain hydrogen bonds
formed between –N–H groups and –C=O groups that are
four residues back, i.e., –N–H group of a 6th peptide bond
is hydrogen bonded to –C=O group of 2nd peptide bond
(Fig).

Fig. 3.2 (a) Right
handed α-helix
(b) Intra chain
hydrogen bonds
between N–H
groups and C = O
groups that are four
residues back

only the atoms on the parts of the coils facing you are shown. If you try
to show all the atoms, the whole thing gets so complicated that it is
virtually impossible to understand what is going on.
Hydrogen bonds

6. Each peptide bond participates in the hydrogen bonding.
This gives maximum stability to α-helix.
7. α-helix present in most fibrous proteins is right handed.
The right handed α-helix is more stable than the left handed
helix.
8. α-helix is hydrophobic in nature because of intra chain
hydrogen bonds.
9. An α-helix forms spontaneously since it is the most stable
conformation of polypeptide chain.
10. Some amino acids act as terminators for α-helix.
Example: Proline.
11. Aromatic amino acids stabilizes α-helix.
12. Charged and hydrophobic amino acids destabilize α-
helix.
13. Content of α-helix varies from protein to protein.

• In a β pleated sheet, two or more segments of
a polypeptide chain line up next to each other,
forming a sheet-like structure held together by
hydrogen bonds.
• The hydrogen bonds form between carbonyl and amino
groups of backbone, while the R groups extend above
and below the plane of the sheet .
• The strands of a β pleated sheet may be parallel,
pointing in the same direction (meaning that their N-
and C-termini match up), or antiparallel, pointing in
opposite directions (meaning that the N-terminus
of one strand is positioned next to the C-terminus of the
other).

β-Pleated Sheet Features
1. In β-pleated sheet, the polypeptide chain is fully
extended.
2. In β-pleated sheet, polypeptide chains line up side by
side to form sheet (Fig). The side chains are above or below
the plane of the sheet.
3. From 2 to 5, adjacent strands of polypeptides may
combine and form these structure.
4. When the adjacent polypeptide chains run in same
direction (N to C terminus) the structure is termed as
parallel β-pleated sheet. (Fig)

5. When the adjacent polypeptide chains run in opposite
direction the structure is termed as anti-parallel β-pleated
sheet (Fig).
6. The β-pleated sheet is stabilized by inter chain hydrogen
bonds (Fig).
7. β-keratin contains anti parallel β-pleated sheet.
8. Both parallel and anti-parallel β-pleated sheet occur in
other proteins. Amyloid protein present in Alzheimer’s
disease has anti parallel β-pleated sheet. It accumulates in
the CNS.

Fig. 3.3 β-Pleated sheet showing arrangement of ‘R’ groups.
All the R-groups project above (solid line) or below (broken
line) the plane

Fig. 3.4 (a) Parallel β-structure showing inter chain hydrogen
bonds

Fig. 3.4 (b) Anti-parallel β-structure

Random Coil (Disordered) Conformation
• Regions of proteins that are not organized as helices and
pleated sheet are said to be present in random coil
conformation.
• These are also equally important for biological function
of proteins as those of helices and β-pleated sheet.
β-turn or β-bends (Reverse Turn)
• Hair pin turn of a polypeptide chain is called as β-turn.
The change in the direction of a polypeptide chain is
achieved by β-turn.
• β-turn connects anti parallel β-sheets. Usually four
amino acids make up β-turn. Gly, Ser, Asp, proline are
involved in β-turns. (Fig. 3.5a)

Super Secondary Structure
In some globular proteins regions of α-helix and β-pleated
sheet join to form super secondary structure or motifs.
They are very important for biological function. (Fig. 3.5b)
Super Helix
α-keratin consist of right handed α-helix as basic unit. Three
such α-helices get cross linked by disulfide bonds and form
super secondary structure. (Fig. 3.5c)
Triple Helix
• Collagen present in skin, cartilage, bone and tendons
consists of left handed helix as basic unit.
• Three left handed helices are wrapped around each
other to right handed super secondary structure triple
helix.

Fig. 3.5 (a) Two anti-parallel chains are joined by β-turn
(b) Motif (c) Super secondary structure

3. Tertiary Structure
• Three-dimensional folding of polypeptide chain is called as
tertiary structure.
• It consists of regions of α-helices, β-pleated sheet, β-turns,
motifs and random coil conformations.
• Interrelationships between these structures are also a part
of tertiary structure (Fig. 3.6).
• Tertiary structure of a protein is mainly stabilized by non-
covalent bonds

The tertiary structure of proteins
What is tertiary structure?
• The tertiary structure of a protein is a description of the
way the whole chain (including the secondary structures)
folds itself into its final 3-dimensional shape.

• The overall three-dimensional shape of an entire protein
molecule is the tertiary structure.
• The protein molecule will bend and twist in such a way as
to achieve maximum stability or lowest energy state.
• Although the three-dimensional shape of a protein may
seem irregular and random, it is fashioned by many
stabilizing forces due to bonding interactions between the
side-chain groups of the amino acids.

• The model shows the alpha-helices in the secondary
structure as coils of "ribbon".
• The beta-pleated sheets are shown as flat bits of ribbon
ending in an arrow head.
• The bits of the protein chain which are just random coils
and loops are shown as bits of "string".
• The tertiary structure of a protein is held together by
interactions between the side chains - the "R" groups.
There are several ways this can happen.

1. Ionic interactions
• Some amino acids (such as aspartic acid and glutamic
acid) contain an extra -COOH group. Some amino acids
(such as lysine) contain an extra -NH2 group.
• Transfer of a hydrogen ion from the -COOH to the -NH2
group to form zwitterions just as in simple amino acids.
• We get an ionic bond between the negative and
the positive group if the chains folded in such a way that
they were close to each other.

2. Hydrogen bonds
• The hydrogen bonds between side groups - not between
groups actually in the backbone of the chain.
• Lots of amino acids contain groups in the side chains
which have a hydrogen atom attached to either an
oxygen or a nitrogen atom. This is a classic situation
where hydrogen bonding can occur.
• For example, the amino acid serine contains an -OH
group in the side chain. You could have a hydrogen bond
set up between two serine residues in different parts of
a folded chain.

• Imagine similar hydrogen bonding involving:
- OH groups, or -COOH groups, or -CONH2 groups, or -NH2
groups in various combinations –
• Also to remember that a -COOH group and an -NH2 group
would form a zwitterion and produce stronger ionic
bonding instead of hydrogen bonds.

3. van der Waals dispersion forces
• Several amino acids have quite large hydrocarbon groups
in their side chains.
• Temporary fluctuating dipoles in one of these groups
could induce opposite dipoles in another group on a
nearby folded chain.
• The dispersion forces set up would be enough to hold
the folded structure together.
4. Sulphur bridges
• The formation of disulfide bridges by oxidation of the
sulfhydryl groups on cysteine is an important aspect of
the stabilization of protein tertiary structure, allowing
different parts of the protein chain to be held together
covalently.

• Under physiologic conditions, the hydrophobic side-chains
of neutral, non-polar amino acids such as phenylalanine or
isoleucine tend to be buried on the interior of the protein
molecule thereby shielding them from the aqueous
medium.
• The alkyl groups of alanine, valine, leucine and isoleucine
often form hydrophobic interactions between one-another,
while aromatic groups such as those of phenylalanine and
tyrosine often stack together.
• Acidic or basic amino acid side-chains will generally be
exposed on the surface of the protein as they are
hydrophilic.

The formation of disulfide bridges by oxidation of the sulfhydryl groups on
cysteine is an important aspect of the stabilization of protein tertiary structure,
allowing different parts of the protein chain to be held together covalently.
Additionally, hydrogen bonds may form between different side-chain groups. As
with disulfide bridges, these hydrogen bonds can bring together two parts of a
chain that are some distance away in terms of sequence. Salt bridges, ionic
interactions between positively and negatively charged sites on amino acid side
chains, also help to stabilize the tertiary structure of a protein.

Quaternary Structure
• Many proteins are made up of multiple polypeptide
chains, often referred to as protein subunits.
• These subunits may be the same (as in a homodimer) or
different (as in a heterodimer).
• The quaternary structure refers to how these protein
subunits interact with each other and arrange
themselves to form a larger aggregate protein complex.
• The final shape of the protein complex is once again
stabilized by various interactions, including hydrogen-
bonding, disulfide-bridges and salt bridges.
• The four levels of protein structure are shown in Figure
2.

The Importance of correct folding
• The primary structure conveys all the information
necessary to produce the correct tertiary structure, but
the folding process in vivo can be a bit trickier.
• In the protein – dense environment of the cell, protein
may begin to fold incorrectly as they are produced, or
they may begin to associate with other proteins before
completing their folding process.
• Correctly folded proteins are usually soluble in the
aqueous cell environment , or they are correctly attached
to membranes.
• However, when proteins do not fold correctly, they may
interact with other proteins and form aggregates (fig).

• This occurs because hydrophobic regions that should be
buried inside the protein remain exposed and interact
with other hydrophobic regions on other molecules.
• Several neurodegenerative disorders, such as
Alzheimer’s, Parkinson’s and Huntington’s diseases are
caused by accumulation of protein deposits from such
aggregates.
• Protein –Folding Chaperones
• To help avoid the protein misfolding problem, special
proteins called chaperones aid in the correct and timely
folding of many proteins.

• The first such proteins discovered were a family called
hsp70 (70,000MW heat-shock protein), which are
proteins produced in E.coli grown above optimal
temperatures.
• Chaperones exist in organisms from prokaryotes through
humans, and their mechanisms of action are currently
being studied.
• It is becoming more and more evident that protein
folding dynamics are crucial to protein function in vivo.

Denaturation
• It is a process in which proteins or nucleic acids lose the
quaternary structure, tertiary structure and secondary
structure which is present in their native state, by
application of some external stress or compound such as a
strong acid or base, a concentrated inorganic salt, an
organic solvent (e.g., alcohol or chloroform), radiation or
heat.
• If proteins in a living cell are denatured, this results in
disruption of cell activity and possibly cell death.
• Protein denaturation is also a consequence of cell death.

• Denatured proteins can exhibit a wide range of
characteristics, from conformational change and loss of
solubility to aggregation due to the exposure of
hydrophobic groups.
• Denatured proteins lose their 3D structure and therefore
cannot function.
How denaturation occurs at levels of protein structure?
• In quaternary structure denaturation, protein sub-units
are dissociated and/or the spatial arrangement of protein
subunits is disrupted.

Tertiary structure denaturation involves the disruption of:
• Covalent interactions between amino acid side-chains
(such as disulfide bridges between cysteine groups)
• Non-covalent dipole-dipole interactions between polar
amino acid side-chains (and the surrounding solvent)
• Van der Waals (induced dipole) interactions between
nonpolar amino acid side-chains.

• In secondary structure denaturation, proteins lose all
regular repeating patterns such as alpha-helices and
beta-pleated sheets, and adopt a random coil
configuration.
• Primary structure, such as the sequence of amino acids
held together by covalent peptide bonds, is not
disrupted by denaturation.
Reversibility
• In many cases, denaturation is reversible (the proteins
can regain their native state when the denaturing
influence is removed). This process can be called
renaturation.

This understanding has led to the notion that all the information
needed for proteins to assume their native state was encoded in the
primary structure of the protein, and hence in the DNA that codes for
the protein, the so-called "Antinsen's thermodynamic hypothesis.
Protein Denaturation
• Denaturation of a protein generally causes its biological
activity to be lost, as well as its solubility.
• Extremes of pH or temperature, as well as detergents or
other substances that reduce disulfide bonds, can cause
protein denaturation.
• All degrees of structure higher than the primary one are
generally destroyed when a protein (or a nucleic acid) is
denatured. Particular peptide bonds in the primary
structure can also be disrupted with extremes of pH.

• Additionally, tryptophan is typically destroyed in acid
hydrolysis, whereas glutamine and asparagine are
deaminated to their respective acids.
• Amino acids are usually racemized in base hydrolysis
(i.e., they lose their optical activity), and serine and
threonine are destroyed.

Protein Stability
• Due to the nature of the weak interactions controlling
the three-dimensional structure, proteins are very
sensitive molecules.
• The term native state is used to describe the protein in
its most stable natural conformation in situ.
• This native state can be disrupted by a number of
external stress factors including temperature, pH,
removal of water, presence of hydrophobic surfaces,
presence of metal ions and high shear.
• The loss of secondary, tertiary or quaternary structure
due to exposure to a stress factor is called denaturation.
Denaturation results in unfolding of the protein into a
random or misfolded shape.

• A denatured protein can have quite a different activity
profile than the protein in its native form, usually losing
biological function.
• In addition to becoming denatured, proteins can also
form aggregates under certain stress conditions.
• Aggregates are often produced during the manufacturing
process and are typically undesirable, largely due to the
possibility of them causing adverse immune responses
when administered.

Collagen
• Collagen is the main structural protein in the extracellular
space in the various connective tissues in animal bodies.
• As the main component of connective tissue, it is the
most abundant protein in mammals, making up from 25%
to 35% of the whole-body protein content.
• Collagen consists of amino acids wound together to form
triple-helices to form of elongated fibrils.
• It is mostly found in fibrous tissues such as tendons,
ligaments and skin.
• The fibroblast is the most common cell that creates
collagen.
• Gelatin, which is used in food and industry, is collagen.

• There are at least 16 types of collagen, but 80 – 90 percent
of the collagen in the body consists of types I, II, and III.
• These collagen molecules pack together to form long thin
fibrils of similar structure.
• Its fundamental structural unit is a long (300- nm), thin
(1.5-nm-diameter) protein that consists of three coiled
subunits: two α1 chains and one α2.
• Each chain contains precisely 1050 amino acids wound
around one another in a characteristic right-handed triple
helix
• All collagens were eventually shown to contain three-
stranded helical segments of similar structure;

The structure of collagen.
(a) The basic structural unit is a triple-stranded helical
molecule. Each triple-stranded collagen molecule is 300
nm long.
(b) In fibrous collagen, collagen molecules pack together side
by side.
The triple-helical structure of collagen arises from an unusual
abundance of three amino acids:
glycine, proline, and hydroxyproline. These amino acids make
up the characteristic repeating motif Gly-Pro-X, where X can
be any amino acid. Each amino acid has a precise function.

• The side chain of glycine, an H atom, is the only one that
can fit into the crowded center of a three stranded helix.
• Hydrogen bonds linking the peptide bond NH of a glycine
residue with a peptide carbonyl (C═O) group in an
adjacent polypeptide help hold the three chains together.
• The fixed angle of the C – N peptidyl-proline or peptidyl-
hydroxyproline bond enables each polypeptide chain to
fold into a helix with a geometry such that three
polypeptide chains can twist together to form a three-
stranded helix.

• Many three-stranded type I collagen molecules pack
together side-by-side, forming fibrils with a diameter of
50 – 200 nm.
• In fibrils, adjacent collagen molecules are displaced from
one another by 67 nm, about one-quarter of their length
Collagen triple helices are stabilized by hydrogen bonds between residues in different
polypeptide chains, a process aided by the hydroxyl groups of hydroxyprolyl residues. Additional
stability is provided by covalent cross-links formed between modified lysyl residues both within
and between polypeptide chains.

Chemistry
• The collagen protein is composed of a triple helix, which
generally consists of two identical chains (α1) and an
additional chain that differs slightly in its chemical
composition (α2).
• The most common motifs in the amino acid sequence of
collagen are glycine-proline-X and glycine-X-
hydroxyproline, where X is any amino acid other than
glycine, proline or hydroxyproline.

Synthesis
• First, a three-dimensional stranded structure is
assembled, with the amino acids glycine and proline as
its principal components.
• This is not yet collagen but its precursor, procollagen.
Procollagen is then modified by the addition of
hydroxyl groups to the amino acids proline and lysine with
the help of hydroxylase enzymes.
• Because the hydroxylase enzymes that perform these
reactions require vitamin C as a cofactor, a long-term
deficiency in this vitamin results in impaired collagen
synthesis and scurvy.

Amino acids
Collagen has an unusual amino acid composition and
sequence:
• Glycine is found at almost every third residue.
• Proline makes up about 17% of collagen.
• Collagen contains two uncommon derivative amino acids
not directly inserted during translation. These amino
acids are found at specific locations relative to glycine
and are modified post-translationally by different
enzymes, both of which require vitamin C as a cofactor.

Tropocollagen molecule: three left-handed procollagens (red, green, blue) join to form a right
handed triple helical tropocollagen.
• Hydroxyproline derived from proline
• Hydroxylysine derived from lysine - depending on the
type of collagen, varying numbers of hydroxylysines are
glycosylated (mostly having disaccharides attached).

Collagen I formation
1. Inside the cell
1. Two types of alpha chains are formed during translation
on ribosomes along the rough endoplasmic reticulum (RER):
alpha-1 and alpha-2 chains. These peptide chains (known as
preprocollagen) have registration peptides on each end and
a signal peptide.
2. Polypeptide chains are released into the lumen of the
RER.
3. Signal peptides are cleaved inside the RER and the chains
are now known as pro-alpha chains.

4. Hydroxylation of lysine and proline amino acids occurs
inside the lumen. This process is dependent on ascorbic
acid (vitamin C) as a cofactor.
5. Glycosylation of specific hydroxylysine residues occurs.
(Glycosylation occurs by adding either glucose or galactose
monomers onto the hydroxyl groups that were placed onto
lysines, but not on prolines)
6. Triple alpha helical structure is formed inside the
endoplasmic reticulum from two alpha-1 chains and one
alpha-2 chain.
7. Procollagen is shipped to the Golgi apparatus, where it is
packaged and secreted by exocytosis.

2. Outside the cell
1. Registration peptides are cleaved and tropocollagen is
formed by procollagen peptidase.
2. Multiple tropocollagen molecules form collagen fibrils, via
covalent cross-linking (aldol reaction) by lysyl oxidase which
links hydroxylysine and lysine residues. Multiple collagen
fibrils form into collagen fibers.
3. Collagen may be attached to cell membranes via several
types of protein, including fibronectin and integrin.

Three polypeptides coil to form
tropocollagen. Many tropocollagens then
bind together to form a fibril, and many of
these then form a fibre.

Ramachandran plot
A Ramachandran plot (also known as
a Ramachandran diagram or a [φ,ψ]
plot), originally developed in 1963 by
G. N. Ramachandran, C.
Ramakrishnan, and V.
Sasisekharan, is a way to visualize
energetically allowed regions for
backbone dihedral angles ψ against
φ of amino acid
residues in protein structure.

The Ramachandran Plot
• In a polypeptide the main chain N-C alpha and C alpha-C
bonds relatively are free to rotate. These rotations are
represented by the torsion angles phi and psi,
respectively.
• The Ramachandran plot is a way to visualize
energetically allowed regions for backbone dihedral angles
ψ against φ of amino acid residues in protein structure.
• To systematically vary phi and psi with the objective of
finding stable conformations.

• For each conformation, the structure was examined for
close contacts between atoms.
• Atoms were treated as hard spheres with dimensions
corresponding to their van der Waals radii.
• Therefore, phi and psi angles which cause spheres to
collide correspond to sterically disallowed conformations
of the polypeptide backbone.

• In the diagram above the white areas correspond to
conformations where atoms in the polypeptide come
closer than the sum of their van der Waals radi. These
regions are sterically disallowed for all amino acids except
glycine which is unique in that it lacks a side chain.
• The red regions correspond to conformations
where there are no steric clashes, ie these are the allowed
regions namely the alpha-helical and beta-sheet
conformations.
• The yellow areas show the allowed regions if slightly
shorter van der Waals radi are used in the calculation, ie
the atoms are allowed to come a little closer together.
This brings out an additional region which corresponds to
the left-handed alpha-helix.

The oxygen has a partial
negative charge and the nitrogen a partial positive
charge, setting up a small electric dipole. The six atoms
of the peptide group lie in a single plane, with the oxygen
atom of the carbonyl group and the hydrogen atom
of the amide nitrogen trans to each other. From these
findings Pauling and Corey concluded that the peptide
C-N bonds are unable to rotate freely because of their
partial double-bond character. Rotation is permitted
about the N-C and the C-C bonds. The backbone
of a polypeptide chain can thus be pictured as a series
of rigid planes with consecutive planes sharing a common
point of rotation at C (Fig. 4–2b). The rigid peptide
bonds limit the range of conformations that can be
assumed by a polypeptide chain.

• By convention, the bond angles resulting from rotations
at C are labeled (phi) for the N-C bond
and (psi) for the C-C bond.
• Again by convention, both and are defined as 180 when
the polypeptide is in its fully extended conformation and
all peptide groups are in the same plane.
• In principle, ψ and φ can have any value between +180
and -180, but many values are prohibited by steric
interference between atoms in the polypeptide
backbone and amino acid side chains.
• The conformation in which both ψ and φ are 0 is
prohibited for this reason; this conformation is used
merely as a reference point for describing the angles of
rotation.

• Allowed values for and are graphically revealed when
is plotted versus in a Ramachandran plot, introduced by
G. N. Ramachandran.

Difference between globular protein and
fibrous proteins

• A Globular protein
1. is spherical in shape and has the property of forming
colloids with water.
• It gets dissolved in water. Globular proteins are also
called as spheroproteins owing to their shape.
Hemoglobin is an example of globular protein.
• Fibrous proteins
1. Fibrous proteins are elongated strand-like structures and
are usually present in the form of rods or wires.
• They are also called as scleroproteins. Keratin, collagen
and elastin are all fibrous proteins. Keratin is found in
hair, horns, nails, feathers etc.

2. An important differentiating feature is that fibrous
proteins are insoluble in water, weak acids and
weak bases but soluble in strong acids and alkalis
whereas globular proteins are soluble in water, acids and
bases.
3. The peptide chains are bound together by strong
intermolecular hydrogen bonds in fibrous proteins whereas
in globular proteins they are held together by weak
intermolecular hydrogen bonds.

4. Fibrous proteins have primary and secondary structures.
They are made up of a single unit or structure which is
repeated multiple times. Fibrous proteins are highly resistant
to digestion by enzymes and are extremely tensile.
Globular proteins are made up of not only primary,
secondary but also tertiary and occasionally quaternary
structures. Globular proteins consist of straight chains of
secondary structures which abruptly join polypeptide chains
and change directions.

Difference in functions
• Globular proteins have multiple functions as they are
used to form enzymes, cellular messengers, amino acids
but fibrous proteins act only as structural proteins.
• Globular proteins are highly branched or coiled structures
and are majorly responsible for transportation of vital
nutrients like oxygen through hemoglobin.
•
• Globular proteins are the major source of hemoglobin,
immunoglobins, insulin and milk-protein casein.
• They also are involved in the formation of amino acids
which are basic building blocks of all proteins.

• They are needed for the formation of chemical
messengers like hormones in the body.
• They are essential for the formation of transporters of
other particles through the membrane.
• Myoglobin is another example of globular protein which
is the chief protein found in muscles.

• Fibrous proteins are needed for the formation of tough
structures like connective tissue, tendons and fibers of
the muscle.
• Collagen is a major component of all our connective
tissues. Fibroin is a fibrous protein which is used to
produce silk by silkworms and webs of spider.
• Fibrous proteins are responsible for the production of
the movements of the muscles and tendons at a joint.
• Hemoglobin
• Hemoglobin is a tetramer, consisting of four polypeptide
chains, two alpha chains, and two beta chains. The two
alpha chains of hemoglobin are identical, as are the two
beta chains. The overall structure of hemoglobin is in
Greek letter notation. The alpha chain is 141 residues
long, and the beta chain is 146 residues long.

Summary:
• Fibrous proteins and globular proteins differ in size,
shape, solubility, appearance as well as in function.
• Fibrous proteins consist of repetition of a single unit to
form chains that act as connective tissues and give
strength and joint mobility.
• Globular proteins are spherical in shape and consist of
long chains with numerous branches and offshoots which
make them great as transport proteins.
• Examples of fibrous proteins are collagen, elastin, keratin,
silk, etc.
• Examples of globular protein are myoglobin, hemoglobin,
casein, insulin, etc.

Chaperones
• 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 subsequent coalescence
into a molten globule.
• The hsp60 family of chaperones, sometimes called
chaperonins.

• 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.

Module-6-Proteins.pdf

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