Chapter 3B:
Proteins, Structure
and Fuction
Textbook of
BIOCHEMISTRY
for Medical Students
By DM Vasudevan, et
al. TENTH EDITION
Revised Reprint
Specific Learning Objectives
The learner will be able to:
•Describe peptide bond formation and properties
•Describe the primary structure of proteins
•Mention the features of secondary, tertiary and quaternary
structures
•Enumerate the steps in sequence analysis (study of primary
structure)
The word protein is derived from Greek word, “proteios”
which means primary. Proteins are of paramount importance
for biological systems.
Out of the total dry body weight, 3/4ths are made up of proteins.
Proteins are used for the body building; all the major structural and
functional aspects of the body are carried out by protein molecules.
Abnormality in protein structure will lead to molecular diseases
with profound alterations in metabolic functions.
Proteins contain Carbon, Hydrogen, Oxygen and Nitrogen as
the major components. Sulphur and Phosphorus are minor
constituents.
Nitrogen is characteristic of proteins. The nitrogen content of
ordinary proteins is 16% by weight.
All proteins are polymers of amino acids.
Peptide bond formation
Proteins
PEPTIDE BOND
R2
H2N CH-CO-NH-CH-COOH
R1
1st amino acid 2nd amino acid
The peptide bond is a partial double bond.
The C–N bond is ‘trans‘ in nature and there is no freedom of
rotation
The distance is 1.32 Å which is midway between single bond (1.49
Å) and double bond (1.27Å).
Characteristics of Peptide Bond
• Partial double bond
• C-N bond is “trans” in nature
• No freedom of rotation (due to partial double bond nature)
• Side chains are free to rotate
• The angles of rotation known as Ramachandran angles,
determine the spatial orientation of the peptide chain
Rigid
The side chains are free to rotate on either side of the peptide bond.
Numbering of Amino Acids in Proteins
• In a polypeptide chain, at one end there will be one free
alpha amino group.
• This end is called the amino terminal (N-terminal) end and the
amino acid contributing the alpha-amino group is named as the
first amino acid.
• Biosynthesis of the protein starts from the amino terminal end.
• The other end of the polypeptide chain is the carboxy
terminal end (C-terminal), where there is a free alpha
carboxyl group which is contributed by the last amino acid.
• All other alpha amino and alpha carboxyl groups are involved in
peptide bond formation.
Dipeptide (2 amino acids)
Tri peptide (3) (ex: Glutathione)
Oligo peptide (5-10)
Poly peptide (10-50)
Protein (>50)
Di peptide (2 amino acids)
Tri peptide (3) (ex: Glutathione)
Oligo peptide ( >10 )
Poly peptide
Protein
Branched and Circular Proteins
• Generally, the polypeptide chains are linear.
• However, branching points in the chains may be produced by
interchain disulphide bridges.
• The covalent disulphide bonds between different polypeptide
chains in the same protein (interchain) or portions of the same
polypeptide chain (intrachain) are also part of the primary
structure.
Pseudopeptides
• Rarely, instead of the alpha COOH group the gamma
carboxyl group of glutamic acid may enter into peptide
bond formation, e.g. Glutathione (gamma-glutamyl-cysteinyl-
glycine).
• The term pseudopeptide (or isopeptide) is used to denote such a
peptide bond formed by carboxyl group, other than that present
in alpha position.
• Very rarely, protein may be in a circular form, e.g. Gramicidin.
Definitions of Levels of Organization
1. Primary structure of protein means the order of amino
acids in the polypeptide chain and the location of disulfide
bonds, if any.
2. Secondary structure is the steric relationship of amino acids,
close to each other.
3. Tertiary structure denotes the overall arrangement and
interrelationship of the various regions, or domains of a single
polypeptide chain.
4. Quaternary structure results when the proteins consist of two
or more polypeptide chains held together by noncovalent forces.
Organisation of Oroteins
Primary
Secondary
Tertiary
Quaternary
Primary Structure = Number and
Unique Sequence of Amino Acids
GLY--ALA--VAL GLY--VAL--ALA
1 2 3 1 2 3
Protein A Protein B
In a tripeptide, there are 3 amino acids, but these 3 can be any
of the total 20 amino acids.
Thus 203 = 8000 different permutations and combinations are
possible in a tripeptide.
An ordinary protein having about 100 amino acids, will have 20100
different possibilities.
By changing the sequence of combination of 20 amino acids, nature
produces enormous number of markedly different proteins.
INSULIN Primary Structure
Primary Structure of Insulin
• Insulin has two polypeptide chains.
• A chain (Glycine chain) has 21 amino acids
• B (Phenyl alanine) chain has 30 amino acids.
• They are held together by two interchain disulphide bonds.
• A chain 7th cysteine and B chain 7th cysteine are connected.
Primary Structure of Insulin
• Similarly, A chain 20th cysteine and B chain 19th cysteine
are connected.
• There is another intrachain disulphide bond between 6th and
11th cysteine residues of A chain.
• Species variation is restricted to amino acids in position 8, 9 and
10 in A chain and in C-terminal of B chain.
• Amino acid sequence has been conserved to a great extent during
evolution.
Conversion of Pro-insulin to active Insulin
Arrows show the site of action of proteolytic enzymes.
A protein with a specific primary structure, when put in
solution, will automatically form its natural three dimensional
shape.
Primary structure determines other levels of organisation
Mutation
B chain 6th Glu --> Val
HbA --> HbS
Structure - function relationship
Secondary structure
Configurational relationship between adjacent 3 - 4 amino acid
residues
Tertiary structure
Relationship of amino acids in three-dimensional rganisation
Primary structure is important
Forces Keeping the Structure
Primary -- Peptide linkage
Disulphide linkage
Secondary and Tertiary
Hydrogen bonds
Electrostatic (ionic)
Hydrophobic bonds
van der Waals force
Hydrogen Bond
Hydrogen releasing
-NH (imidazole, indole, peptide)
-OH (Serine, Threonine)
-NH2 (Arginine, Lysine)
Hydrogen accepting
COO- (Aspartic, Glutamic)
C=O (peptide)
Electrostatic or ionic bonds
+ ve : Lysine, Arginine, Histidine
- ve : Aspartic, Glutamic
Hydrophobic Bonds
Non-polar hydrobic side chains
VLIMPW
Van der Waals Forces
• Attractive forces operating between all atoms due to
oscillating dipoles
• Dependent on distance between atoms
• Weak forces
• Contributes maximum towards stability of protein
Alpha Helix
• Most common and stable conformation; e.g.,
Hemoglobin
1. Right handed; Spiral
structure.
2. Polypeptide bonds
backbone
Alpha Helix
3. Structure is stabilized by
hydrogen bonds between
NH and C=O groups.
4. Each turn by 3.6 residues;
5. Distance between each
residue is 0.15 nm
6. Side chains of amino acids extend outward.
Beta-pleated Sheet
• Polypeptide chains fully extended.
• Stabilised by hydrogen bonds between NH and C=O groups of
neighbouring polypeptide segments.
• Intrachain disulfide bridges stabilise these bends.
• Adjacent strands in a sheet can run in the same direction
(parallel) or in opposite direction (anti parallel sheet)
• Silk Fibroin (anti parallel)
• Flavodoxin (parallel)
• Carbonic anhydrase (both)
Structure of Beta-pleated Sheet
Three dimensional
structure of myoglobin
The tertiary structure denotes
three dimensional structure of
the whole protein
The tertiary structure is
maintained by hydrophobic
bonds, electrostatic bonds and
van der Waals forces. The
tertiary structure acquired by
native protein is always
thermodynamically most
stable.
Forces Keeping the Tertiary Structure
Domain
• Domain is the term used to denote a compact globular
functional unit of a protein.
• A domain is a relatively independent region of the protein, and
may represent a functional unit.
• The domains are usually connected with relatively flexible areas
of protein.
• Phenyl alanine hydroxylase enzyme contains 3 domains, one
regulatory, one catalytic and one protein-protein interaction
domains.
Motif
In a protein, a structural motif is a superstructure. Different
structural motifs are:
a) Beta hairpin: This is very common. Two antiparallel beta-
strands are connected by a tight turn of a few amino acids
between them.
b) Helix-loop-helix: This consists of alpha-helices bound by
a looping stretch of amino acids. This motif is seen in
transcription factors.
c) Zinc finger: Two beta-strands with an alpha-helix end
folded over to bind a zinc ion. This is important in DNA
binding proteins.
d) Helix-turn-helix: Two alpha-helices joined by a short
strand of amino acids and found in many proteins that
regulate gene expression.
Specific Motifs in proteins
Protein Structural motif present
Myoglobin Alpha-helix and beta-pleated
sheet
Collagen Triple helix
Keratin Coiled coil
Elastin No specific motif
Superoxide dismutase Antiparallel,
beta-pleated sheet
Quaternary Structure
Polypeptides aggregate to form one functional protein.
Loss of function when the subunits are dissociated.
The forces that keep the quaternary structure are hydrogen bonds,
electrostatic bonds, hydrophobic bonds and van der Waals forces.
Quaternary Structure
Subunit or monomer
Combination: Dimer (2) tetramer (4)
Hb: 2 alpha and 2 beta chains
Immunoglobulin: 2 heavy and 2 light
Creatine kinase (CK) = dimer
Lactate dehydrogenase (LDH) = 4
Aspartate transcarbamoylase = 6
Hemoglobin
Configuration and Conformation
Configuration of a protein denotes the spatial relationship
between particular atoms, e.g. L and D amino acids.
Conformation means the spatial relationship of every atom in a
molecule, e.g. rotation of a portion of the molecule.
Levels of Organisation
1. Primary structure of protein means the order of amino
acids in the polypeptide chain and the location of disulfide
bonds, if any.
2. Secondary structure is the steric relationship of amino acids,
close to each other.
3. Tertiary structure denotes the overall arrangement and inter-
relationship of the various regions, or domains of a single
polypeptide chain.
4. Quaternary structure results when the proteins consist of two
or more polypeptide chains are held together by non-covalent
forces.
Structure-Function Relationship
• The three dimensional structural conformation provides
and maintains the functional characteristics.
• The three dimensional structure, in turn, is dependent on the
primary structure.
• Any difference in the primary structure may produce a protein
which cannot serve its function.
Examples
1. Enzymes - The first step in enzymatic catalysis is the
binding of the enzyme to the substrate. This, in turn,
depends on the structural conformation of the active site of the
enzyme, which is precisely oriented for substrate binding.
2. Hemoglobin - Binding of oxygen to one heme facilitates oxygen
binding by other subunits. Even a single amino acid substitution
alters the structure and thereby the function.
3. Collagen - Collagen forms a superhelical cable where the
3 polypeptide chains are wound around itself.
• In collagen, every 3rd residue is a glycine.
• The only amino acid that can fit into the triple stranded helix is
glycine.
• Triple helix of collagen is stabilized by the steric repulsion of the
rings of hydroxyproline and also by the hydrogen bonds between
them.
Proteins
Study of Protein Structure
Sequence analysis
Complete Hydrolysis
6 N HCl; 110 C; 18-36 hr ; anaerobic conditions
No. of polypeptide chains
Dansyl Chloride
N-terminal amino acid
Partial Hydrolysis
Trypsin
Carboxyl group of Arg / Lys
G--A--K-||-M--V--R-||-G--V—
Chymotrypsin
End Group Analysis Dansyl Chloride
H3C N-CH3
Alpha amino group SO2Cl
N-terminal amino acid Dansyl Derivative
End Group Analysis Sanger’s Reagent
NO2
F NO2
Fluoro dinitro benzene (FDNB) Dinitro phenyl derivative
Edman’s Degradation
Phenyl iso thio cyanate
N C=S
Amino group of First a.a.
Label first amino acid
Release
Steps in Edman's degradation process.
Label first amino acid
Release
Label next amino acid
Release
Steps in Edman's degradation process.
Carboxy Peptidase
Sequential release of amino acids from C-terminus
1-----2------3-----4----5
1-----2------3-----4 5
1-----2------3 4 5
1-----2 3 4 5
Ingram’s Technique Protein Finger Printing
Protein digested by Trypsin
Mixture of peptides separated
by chromatography
Peptide mapping
Mutation
position of peptide is altered
Peptide mappings of
Hb in HbA and HbS
are different
NORMAL ABNORMAL
