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Proteins
2.1 INTRODUCTION
The term protein is derived from the greek work proteios meaning “of prime importance”
or “primary” or “holding first place” with respect to the cell constituents. Proteins are the
most abundant organic molecules in the cells constituting 50% or more of their dry
weight. They are found in every part of the cell, as they are fundamental in all aspects of
cell structure and function. There are a huge variety of proteins, each specialised for a
different biological function.
Amino acids are the building blocks of proteins. An amino acid consists of amino
group, a carboxyl group, hydrogen atom and a functional R-group, which are bonded to an
α-carbon atom. In proteins, the α-amino group of one amino acid is joined to the
α-carboxyl group of another amino acid by a peptide bond. All proteins, whether obtained
from the bacteria or from the most complex form of life, contain only a set of 20 amino
acids, called standard amino acids.
2.2 CLASSIFICATION
Proteins are classified based on their composition, function, and conformation or structure.
2.2.1 Classification Based on Composition
Proteins are divided into two major classes on the basis of their composition – Simple
proteins and Conjugated proteins.
Simple proteins are those which on hydrolysis yield only amino acids and no other
major organic or inorganic hydrolysis products. They usually contain about 50% carbon,
7% hydrogen, 23% oxygen, 16% nitrogen and 0–3% sulfur.
Conjugated proteins are those which on hydrolysis yield not only amino acids but also
organic or inorganic components. The non-amino acid part of a conjugated protein is
called prosthetic group. Conjugated proteins are classified on the basis of the chemical
nature of their prosthetic groups.
16 Comprehensive Biotechnology–III
Class
Prosthetic group
Example
Lipoprotein
Glycoprotein
Phosphoprotein
Hemoprotein
Flavoprotein
Metalloprotein
Lipid
Carbohydrate
Phosphate group
Heme (Iron Porphyrin)
Flavin nucleotides
Iron
Zinc
Calcium
Copper
Molybdenum
RNA
β-lipoprotein of blood
Immunoglobulin G
Casein of milk
Hemoglobin
Succinic dehydrogenase
Ferritin
Alcohol dehydrogenase
Calmodulin
Plastocyanin
Dinitrogenase
Ribosome
Nucleoprotein
2.2.2 Classification Based on Biological Function
Proteins have many different biological functions, and are classified based on their function.
Types and Examples
Function
Enzymes
Hexokinase
Lactate dehydrogenase
Phosphorylates glucose
Oxidises lactate
Storage proteins
Ovalbumin
Casein
Gliadin
Zein
Egg-white protein
Milk protein
Seed protein of wheat
Seed protein of corn
Contractile proteins
Myosin
Actin
Thick filaments in myofibril
Thin filaments in myofibril
Transport proteins
Hemoglobin
Myoglobin
Serum albumin
Lipoprotein
Transports
Transports
Transports
Transports
oxygen in blood of vertebrates
oxygen in muscle cells
fatty acids in blood
lipids in blood
Hormones
Insulin
Adrenocorticotropic hormone
Regulates glucose metabolism
Regulates corticosteroid synthesis
(Contd.)
Proteins 17
Types and Examples
Function
Protective proteins in vertebrate blood
Antibodies
Fibrinogen
Thrombin
Form complexes with foreign proteins
Precursor of fibrin in blood clotting
Component of clotting mechanism
Toxins
Ricin
Diphtheria toxin
Clostridium botulinum toxin
Toxic protein of castor bean
Bacterial toxin
Causes bacterial food poisoning
2.2.3 Classification Based on Structure
In the native state, each type of protein molecule has a characteristic three-dimensional
structure, referred to as its conformation. Depending on their conformation, proteins are
classified as Fibrous and Globular Proteins.
Fibrous proteins consist of polypeptide chains arranged in parallel along a single axis
to yield long fibers or sheets. Fibrous proteins are insoluble in water or dilute salt
solutions. They are the structural elements in the connective tissue of higher animals.
For example, collagen of tendons and bone matrix, elastin of elastic connective tissue, αkeratin of hair, horn, skin, nails, feathers, etc.
Globular proteins consist of polypeptide chains tightly folded into compact spherical
or globular shapes. Most globular proteins are soluble in aqueous solutions. They have a
mobile or dynamic function in the cell. Of the nearly 2000 different enzymes known todate, nearly all are globular proteins.
Some proteins fall between the fibrous and globular types, resembling fibrous proteins
in their long rod-like structures and the globular proteins in their solubility in aqueous
salt solutions. For example, myosin, an important structural element of muscle and
fibrinogen, the precursor of fibrin, the structural element of blood clots.
2.3 STRUCTURAL ORGANISATION OF PROTEINS
The function of protein can be understood only in terms of three dimensional structure of
proteins. The structural descriptions of proteins are described in terms of four level of
organisations:
1. Primary structure — the amino acid sequence in the polypeptide chain.
2. Secondary structure — the local spatial arrangement of a polypeptide backbone
atoms, without regard to the arrangement of amino acids side chain. This refers to
the α-helices or β-pleated sheet, and the random coil structure.
3. Tertiary structure — the three dimensional structure of an entire polypeptide
chain (polypeptide backbone and amino acid side chain).
4. Quaternary structure — the spatial arrangement of subunits. Many proteins are
composed of two or more polypeptide chains, loosely referred to as subunits, which
associate through non-covalent interactions, and in some cases covalently associated
through disulphide bonds.
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2.3.1 Primary Structure
Amino acids are the building blocks of proteins. The amino acids are held together in a
protein by covalent bond which are known as peptide bonds or linkages. A peptide bond is
formed by the condensation of the amino group of an amino acid with the carboxyl group
of another amino acid. A dipeptide will have two amino acids, but contains one peptide
bond. Peptides containing more than 10 amino acids are referred to as polypeptides.
O
+
N
C
+
C
Ca
O
C
Ca
N
O
-
O
Alanine
Glycine
O
C
f
N-terminus
N
C
+
N
- C-terminus
C
Ca
O
O
Y Peptide
bond
Ca
Plane of
amide group
+
O
Water
Glycylalanine
The peptide bond has partial
double bond character
O
O
C
N
C
H
Resonant Structures
+
N
H
Proteins 19
Linus pauling and Robert Corey, in late 1930’s analysed the peptide bond. The αcarbon of adjacent amino acid residues are separated by 3 covalent bonds, arranged as –
Cα – C – N – Cα – C – N -. The peptide bond in shorter than the C – N bond in a simple
amine. The atoms associated with peptide are coplanar indicating a resonance structure.
The six atoms of the peptide 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. The peptide
bonds due to partial double bond character are unable to rotate freely. Rotation is permitted about N – Cα (ϕ) and the Cα – C bonds (ψ). Both the – C = O (carbonyl group) and –
NH groups of peptide bonds are polar and are involved in hydrogen bond formation.
The peptide chains are written with the free amino end (N – terminal residue) at the
left, and the free carboxyl end (C – terminal residue) at the right. The amino acid
sequence is read from the N – terminal end to the C – terminal end. The amino acids in
a peptide or protein are represented by the three-letter or one-letter abbreviation.
The amino acid sequence in the polypeptide chain represents the primary structure.
The bovine polypeptide hormone, insulin, was the first protein for which complete amino
acid sequence was determined by Frederick Sanger in 1953. The elucidation of the
primary structure of 51 amino acid residue, insulin, was the labor of many scientists over
the period of a decade, and they utilised ~ 100 gm protein.
Insulin consists of two chains, linked by disulfide bridges. Chain A has 21 amino acid
residues while chain B has 30 amino acid residues. The two chains are bound together in
their quaternary structure by two disulphide bridges. In addition, a third disulphide
bridge between two amino acids in the “A” chain help stabilise its tertiary structure.
30
Ala.Lys.Pro.Thr.Tyr.Phe.Phe.Gly
Arg
NH2 NH2
Chain B
Glu
1
Phe.Val.Asp.Glu.His.Leu.Cys.ly.Ser.His.Leu.Val.Glu.Ala.Leu.Tyr.Leu.Val.Cys.Gly
S
Chain A
S
NH2
S
NH 2
NH2 S
NH2
1
21
Gly.Ile.Val.Glu.Glu.Cys.Cys.Ala.Ser.Val.Cys.Ser.Leu.Tyr.Glu.Leu.Glu.Asp.Tyr.Cys.Asp
S
S
2.3.2 Secondary Structure
The secondary structure refers to the local conformation of its backbone. It is the precise
and repeating folding due to hydrogen bonding with respect to the amino acid backbone
into a helix or a β-pleated sheet and turns and random coil structure. The most common
structure is the α-helix and β-pleated sheet. This structure is a pleated sheet formed by
parallel chains of amino acids. These sheets are important in many structural proteins.
Many proteins have sheets and helices. Secondary structure arises from the geometry of
the bond angle between amino acids as well as hydrogen bonds between nearby amino
acids.
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2.3.2.1 Helical Structure
The polypeptide chain is twisted by the same amount about each of its Cα atoms and the
chain assumes a helical conformation. A helix is characterised by the number, n, of
peptide units per helical turn, and its pitch, p — the distance, the helix rises along its axis
per turn. The helix has chirality, and it may be either right-handed or left-handed. The
helical structure is stabilised by hydrogen bonds. Pauling and Corey in 1951 discovered
the α-helix (Nobel Prize, 1954) through model building. The polypeptide backbone is
tightly wound around an imaginary axis drawn longitudinally through the middle of the
helix and the R-groups of the amino acid residues protrude outward from the helical
backbone. They studied the flexibility in the covalent bonds of a peptide backbone structure as well as the partial charges of the backbone structure, and determined that
peptides could bend EVERY FOURTH (actually every 3.4) amino acid (alpha helix). Alpha
means it twists in a right handed (clockwise manner). The α-helix is the predominant
structure in α-keratins, such as hair.
Secondary structure (helix)
A typical growth rate for human hair is about one half of an inch per month. This
requires that the hair follicle produce approximately ten turns of alpha helical protein
every second. Ten turns, by the way, is about thirty-six amino acids. The protein strands
within hair and other alpha-keratins are crosslinked to some extent by covalent bonds
between cysteine residues to form disulphide bonds. The more such disulphide bonds
there are between the strands, the more rigid the protein becomes as a whole. The alpha
keratins can be classed as “soft” or “hard” according to their sulphur content, which is to
Proteins 21
say, the relative number of cysteines in the polypeptide chains. The low-sulphur keratins
of skin and callous are much more flexible than the high-sulphur, hard keratins of horns,
claws and hooves.
Cysteine-Cystine Transformation
The basic principle of the permanent wave process for hair involves breaking the existing
disulphide bonds between α-helices and then reforming new disulphide bonds after the
hair fibers have been shaped and rearranged by the hair stylist. A reducing agent is first
applied to the hair to break the disulphide bridges. The hair is then arranged into the
desired shape and an oxidising agent applied to cause reformation of the disulphide
bonds. A “permanent” is really only permanent for the portion of the hair that was
processed, and it lasts until new, untreated keratin replaces it.
C
C
H
H
C
N
H
O
H
O
H
N
C
H
H
C
N
H
H
N
H
C
H
S
H
S
S
H
S
C
N
H
C
H
HO
N
O
2 Cysteine
H
+ 2H
H
C
N
H
C
N
+
H
HO
O
H
Cystine
2.3.2.2 β-Pleated Sheet
Linus Pauling and Robert Corey postulated the existence of a second type of secondary
structure, the β-pleated sheet. This conformation utilises the full hydrogen bonding
capacity of the polypeptide backbone, but the hydrogen bonding occurs between the
neighbouring polypeptide chains rather than within the same polypeptide chain as in the
case of α-keratin. β-pleated sheets are of two types:
1. The antiparallel β-pleated sheet, in which neighbouring hydrogen bonded polypeptide chains run in opposite directions
2. The parallel β-pleated sheet, in which neighbouring hydrogen bonded polypeptide
chains extend in the same direction
β-pleated sheets are common structural motifs in proteins. In globular proteins, they
consist of 2 to as many as 15 polypeptide strands, the average being 6 strands. Each
polypeptide chain in a β-sheet contains upto 15 amino acid residues, with the average
being 6 amino acid residues. Parallel β-sheets of less than 5 strands are rare and are less
22 Comprehensive Biotechnology–III
stable than antiparallel β-sheets. This is due to the distortion of hydrogen bonds in
parallel β-sheets in comparison to those of antiparallel β-sheets. Mixed parallel- antiparallel
β-sheets are commonly found in proteins.
Antiparallel β-pleated Structure
N
Ca
Ca
HO
Ca
Ca
C
N
H
O
H
O
H
O
N
C
N
C
C
N
O
Ca
Ca
H
N
C
H
O
Ca
N
C
N
C
H
C
N
C
H
H
H
O
O
N
C
C
N
Ca
OH
Parallel β-pleated Structure
C
N
Ca
N
C
O
H
C
N
Ca
C
N
O
H
Ca
Ca
O
H
H
N
O
H
C
N
C
Ca
H
O
Ca
N
C
N
C
N
H
Ca
C
H
H
Ca
N
Ca
O
O
O
HO
Ca
H
C
HO
N
N
C
O
O
C
H
Ca
HO
Ca
H
N
H
H
C
O
H
O
Proteins 23
2.3.2.3 Random Coil Structure
Regular secondary structures — helices and β sheets comprise around half of the average
globular protein. The remaining polypeptide segment has a loop or coil conformation.
These structures are orderly structures just like helices or sheets. In contrast, random
structure or coil structure refers to the totally disordered and rapidly fluctuating set of
conformations assumed by denatured proteins in solution.
Many proteins have regions that are truly disordered, and often wave around in
solution because there are few forces to hold them in place. Sometimes, entire polypeptide
chain segments are disordered. These may play a role in the binding of specific molecules.
2.3.3 Tertiary Structure
Tertiary structure refers to a higher level of folding in which the helices and sheets of the
secondary structure fold upon themselves. This higher level folding arises for several
reasons. First, different regions of the amino acid chain are hydrophilic or hydrophobic
and arrange themselves accordingly in water. Second, different regions of the chain bond
with each other via hydrogen bonding or disulfide linkages.
Myoglobin is the first protein whose tertiary structure was established by Kendrew
(Nobel Prize 1962) using X-ray diffraction. Most water soluble-proteins have a hydrophobic interior and a hydrophilic exterior.
A common way for enzymes to denature is to unfold either because of hydrogen bond
breakage (often due to pH or temperature), or oxidants or reductants that unnaturally
break or form disulphide bridges. In any case, the active site is affected, which in turn
affects the activity. The final protein structure depends upon how the protein folds as it
comes off of the ribosome and any subsequent processing (i.e., the functional folded
protein may not be the way the protein would fold if it were completely unfolded and
allowed to refold). Sometimes, there are chaperones that help, proteins fold correctly
(after being made off of a ribosome or where, there is a tendency for denaturing).
Chaperones themselves are proteins.
b
Tertiary structure: One complete protein chain
(bchain of hemoglobin)
2.3.4 Quaternary Structure
Quaternary structure arises when different polypeptide chains or subunits are bound
together usually by hydrogen bonds. For example, hemoglobin — the oxygen carrying
24 Comprehensive Biotechnology–III
protein in blood has four subunits hydrogen bonded together. Usually the function of the
total quarternary structure is ‘better’ than the function of the sum of the individual
protein chains. Most proteins with a molecular weight of 50,000 or more are made of such
units.
Sometimes, quaternary structure maybe very complex. For example, beef glutamate
dehydrogenase is an enzyme with a molecular weight of 2,200,000. Each enzyme molecule
consists of eight large subunits. In turn, each of these consists of numerous smaller units.
These polypeptide chains self-assemble into a complete and functional protein. The cell
takes full advantage of this property to rapidly generate the cytoskeleton much of which
consists of very long chains or helices, or tubes of proteins subunits.
Hemoglobin carries oxygen from the lungs to the tissue. Myoglobin performs a similar
function in muscle tissue, taking oxygen from the hemoglobin in the blood and storing it
or carrying it around until needed by the muscle cells. Hemoglobin and myoglobin also
have similar structures. Myoglobin contains 151 amino acid residues plus a heme group
to bond to oxygen. Hemoglobin has four chains, two with 141 residues and a heme group
and two with 146 residues and a heme group. The molecular weight of hemoglobin is
about 64,500 and can carry four oxygen molecules.
It is important that hemoglobin can bond to oxygen under certain conditions. But it is
equally important that hemoglobin can release oxygen under other conditions. The ability
of hemoglobin to bind oxygen is sensitive to several factors. They include pH, temperature,
concentrations of O2 and CO2, and even the number of oxygen molecules already bound.
When oxygen binds to hemoglobin, the structure of the hemoglobin changes slightly so
that it binds more efficiently to oxygen, thus enhancing its ability to carry more oxygen.
b2
a2
b1
a1
Quaternary structure: Four subunits of hemoglobin assembled into
an oligomeric protein