Science of Living System

BS10003
Proteins
Q M
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L T V K

Mainak Bose
School of Bioscience
Email: mainak29@iitkgp.ac.in
 Central Dogma of life
DNA: Storage Medium
Polymer of nucleotides

CCTGAGCCAACTATTGATGAA

RNA: Transmission Medium

Polymer of nucleotides

CCUGAGCCAACUAUUGAUGAA

Protein: Molecular Machines

Polymer of amino acids

PEPTIDE
Proteins come in various shapes and sizes
 Primary Structure of Proteins
The primary structure of a protein is its amino acid sequence
 Amino acids: Building blocks of Proteins
• Protein is a polymer of amino acids.
• There are 20 common amino acids.
• Amino acids have a common
chemical structure - A tetrahedral
sp3 carbon (Cα) with four different
functional groups:
1. Amino group
2. Carboxyl group
3. H-atom
4. Side chain (R) with distinct
chemical property
The 20 Common Amino Acids of Proteins
 Proteins are polypeptide chains
Successive polypeptide bonds: main chain or backbone
Formation of the peptide bond
 The amide plane:
partial double bond character of the peptide bond

• Resonance interactions between electrons in

the C=O bond and the C–N bond of the
peptide group
• Sharing of electrons between these bonds is
an example of Resonance
 Cα atoms are on
opposite sides of the
peptide bond

Cα Cα

Cα Cα
TRANS

H H
Cα Cα
Cα atoms are on the CIS
same side of the
Cα peptide bond Cα
In general, peptide bonds are in the trans conformation
Torsion angles: Φ (phi) and Ψ (psi)

Peptide Plane
Visualizing a few torsion angles

Front Back
1 4 4 (behind 1)
1

2 3
2
3 (behind 2)
 Visualizing a few torsion angles
4 1 Fr Ba 4
1 on ck
0° t
2 3
2
3

1 1 Fr Ba
4 on ck 4 Atom 4 is above
+45° t the plane of the
2 3
screen
 Visualizing a few torsion angles
1 1 Fr Ba
4 on ck 4 Atom 4 is below
-45° t the plane of the
2 3
screen

1 1 Fr Ba
on ck Atom 4 is below
-135° t the plane of the
2 3
screen
4 4
 Ramachandran Plot
G. N. Ramachandran

Φ Ψ
Ψ (psi)

Φ (phi)
 Ramachandran Plot
α-Helix

β-Strand
Ψ (psi)

Φ (phi)
Hierarchy of Protein
Structure
Protein Molecules are Organized in a Structural
Hierarchy

Primary

Secondary

Tertiary

Quaternary
 Secondary Protein Structure
Characterized by main chain NH and CO groups participating in H-bonds

Beta Sheet

Alpha helix
 Alpha Helix

Every 3.6 residues make one turn

The distance (pitch of helix)

between two turns is 5.4 Å

The C=O of residue ‘n’ is hydrogen

bonded to N-H of residue ‘n+4’

n n+1 n+2 n+3 n+4 n+5

The Alpha-Helix has a Dipole Moment

n n+1 n+2 n+3 n+4 n+5

Macro dipole

The dipoles of peptide units are aligned along the

α helical axis
 Alpha Helix: Right-handed or Left-handed?

Alpha helix can be – Right-

handed or Left handed

BUT, left handed helix is not

possible for L-amino acids
due to close approach of
the side chains and CO
group.

Right handed – most

commonly observed in
proteins.
 β-sheet
(Number of β-Strands are Involved)

β-sheet from several regions of the

chain; Each β-strand, typically 5-10 α-helix: from one
residues long continuous region
H-bonds are perpendicular to strands
Parallel and Antiparallel β-sheet

β-pleated sheet: ‘pleated’ because

side chains point up and down
alternatively
Mixed β-sheet
 Polypeptide Chains Fold into Several Domains
• Fundamental unit of tertiary structure – DOMAIN
• Domain: polypeptide chain or a part of polypeptide chain that can
independently fold into a stable tertiary structure
• Domains are also units of function

Tertiary structure refers to the spatial arrangement of amino acid residues

that are far apart in the sequence and to the pattern of disulfide bonds.
 Quaternary Structure
• Proteins containing more than one polypeptide chain exhibit a fourth
level of structural organization. Each polypeptide chain in such a
protein is called a subunit.
• Quaternary structure refers to the spatial arrangement of subunits
and the nature of their interactions.
• The simplest quaternary structure is a dimer, consisting of two
identical subunits.
Quaternary Structure (higher order)

The α2β2 tetramer of human haemoglobin.

The structure of the two identical α subunits
(red) is similar to but not identical with that
of the two β subunits (yellow).

Complex Quaternary Structure. The coat

of rhinovirus comprises 60 copies of each
subunits
 The Protein Folding Problem
Protein folding is the physical process by which a linear polypeptide folds into
its characteristic and functional three-dimensional structure
 φ and ψ torsion angles are the only degrees of
freedom for the backbone
3

1
The protein folding paradox
▪ Consider a small protein with 100 residues.

▪ Cyrus Levinthal calculated that, if each residue can assume

three different conformations, the total number of structures
would be 3100, which is equal to 5 × 1047. If it takes 10-13 s to
convert one structure into another, the total search time would
be 5 × 1047 × 10-13 s, which is equal to 5 × 1034 s, or 1027 years
i.e. longer than the age of the universe!

▪ Clearly, it would take much too long for even a small protein to
fold properly by randomly trying out all possible
conformations.

▪ The enormous difference between calculated and actual

folding times is called Levinthal's paradox.
 The Protein Folding Problem:
Important Questions

1. How do proteins fold? i.e. How do proteins achieve

their final folded structure?
2. How do proteins fold so fast? Most proteins fold within
milliseconds.
3. Can we predict protein structures without
experimentally solving them?
4. Can we design artificial proteins with unique functions
to solve some of our problems?
 The 3D structure of a protein is encoded in its
primary sequence
❑ Protein folding: a set of ordered pathways by which 1D amino
acid sequence of a protein folds into its native functional 3D
conformation.

❑ Thermodynamic hypothesis of Protein Folding: The

interactions between the atoms in a protein control the folding of
the protein molecule into a well-defined three-dimensional
structure.

❑ In 1950s Christian Anfinsen showed that the amino acid

sequence contains enough information required for the proper
folding of the protein into its functional three-dimensional
structure.
 Anfinsen’s Experiment
• Ribonuclease A (RNase A) enzyme
was used for the experiment

• RNase catalyses breakdown of RNA

molecules:
RNase A
– Catalytic Function is a readout of its
3D conformation

• 124 aa long, with 4 disulphide bonds

• Anfinsen treated RNase with:

1. Urea: disrupts all non-covalent bonds
(H-bonds, ionic bonds etc.), leading to
unfolding of RNase

2. BME (β-mercapto ethanol): breaks all

S-S (disulfide) bonds
Disulfide bond forms between two cysteine residues by
reaction between the sulfhydryl (SH) side chains
Anfinsen’s Experiment

1.
2. BME (β mercapto ethanol)
treatment breaks all S-S
bonds
 Anfinsen’s Experiment

If we understand HOW PROTEINS FOLD, we can predict their structure from

sequence! Then we can design proteins with novel functions.
 8 Cys

Select two at a time to form a disulphide bond

8
C2 * 6C2 * 4C2 * 2C2 / 4! = 2520/4! = 105

# of ways to choose the It does not matter in which order

first disulfide bond the FOUR disulphide bonds are
formed. # of permutations of
FOUR disulfide bonds.
How Proteins Fold

❑ Important interactions between amino acids:

• Hydrophobic interactions
• Hydrogen bonding interactions
• Electrostatic interactions

❑ Animation of Protein Folding Funnel:

https://www.youtube.com/watch?v=YANAso8Jxrk
 The Protein Folding Problem
1. How do proteins fold? i.e. How do proteins achieve their final folded
structure?
2. How do proteins fold so fast? Most proteins fold within
milliseconds.
⮚ Protein Folding is a stochastic process i.e. not all conformations are
sampled.
⮚ Proteins fold in small segments (~20 amino acids) independent of
the rest. These folded segments or FOLDONS collapse to give the
final structure.
⮚ Molecular Dynamics (MD) Simulations use physical laws to study
protein folding
The protein folding game - Foldit
https://fold.it
 Important questions on Protein Folding
3. Can we predict protein structure from sequence
• Anfinsen’s experiment demonstrates that a protein sequence encodes its structure.
• Can we decipher this code? I.e. can we predict the structure of a protein from its
primary sequence?
Prediction
Sequence Structure

A C D E
F G H I 1,72,807
K L M N
P Q R S
T V W Y

20 Amino Acids 300 AA/Protein 21,000 Proteins 200,000,000 Proteins

in Human body in the world
 The Protein Folding Problem
3. Can we predict protein structure from sequence
• Anfinsen’s experiment demonstrates that a protein sequence encodes its structure.
• Can we decipher this code? I.e. can we predict the structure of a protein from its
primary sequence? Prediction
Sequence Structure
 Protein misfolding leads to several diseases
Several diseases occur due to misfolding of proteins. Few examples:
1) Cystic Fibrosis: It results from the misfolding of Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR), a protein functioning as a chloride (Cl-) ion
channel. The loss of CFTR function interferes with the body’s ability to efficiently
secrete fluids and salts. It damages the lungs and digestive systems.

https://medlineplus.gov/ency/article/000107.htm Frontiers in Pharmacology 2016 v-7, p-275

 Protein misfolding leads to several diseases
Several diseases occur due to misfolding of proteins. Few examples:
2) Alzheimer’s Disease (AD): It results from the aggregation and precipitation of a
peptide called amyloid-β (Aβ). AD is the most common form of progressive
dementia in the elderly, and of neuro-degenerative diseases in general.

Normal Brain Brain with AD

Comparison of the
two Brains
www.alz.org
 Protein misfolding leads to several diseases
Several diseases occur due to misfolding of proteins. Few examples:
3) Parkinson’s Disease (PD): It results from aggregation and precipitation of the
protein α-synuclein. PD is a motor disorder common among the elderly (but can
also hurt young people). It leads to shaking, stiffness, and difficulty with walking,
balance, and coordination.

http://www.askdrray.com/parkinsons-disease-may-stopped/ Lancet Neurol 2018; 17: 939–53

 Protein misfolding leads to several diseases
Several diseases occur due to misfolding of proteins. Few examples:
4) Creutzfeldt-Jakob Disease (CJD): It is caused by the aggregation and
precipitation of the protein prion. It results in progressive motor dysfunction,
cognitive impairment, and cerebral ataxia.

Higher order
Aggregation Aggregation
Misfolding

Nature Structural & Molecular Biology 19, 370-377 (2012)

 Important Questions on Protein Folding

1. How do proteins fold? i.e. How do proteins achieve their

final folded structure?
2. How do proteins fold so fast? Most proteins fold within
milliseconds.
3. Can we predict protein structures without experimentally
solving them?
4. Can we design artificial proteins with unique
functions to solve some of our problems?
 Protein Design Problem

Protein structure prediction

(for proteins found in nature)
M
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Protein design
(for artificial proteins)
Infection cycle of Influenza Virus
 Designed Protein Targets the Conserved Stem Region
of Influenza Hemagglutinin

Science 2011 32:816

 Methods to study protein structures

X-ray crystallography
• Prof. Amit K Das
(http://www.iitkgp.ac.in/department/BT/faculty/bt-amitk)

Structure-guided protein engineering

• Prof. Dibyendu Samanta
(
http://iitkgp.ac.in/department/BS/faculty/bs-dibyendu.samanta)
NMR spectroscopy
• Nuclear magnetic resonance (NMR) spectroscopy
• Prof. Soumya De
(http://iitkgp.ac.in/department/BS/faculty/bs-somde)
 Protein Structure, Function, Kinetics and
Energetics

Books Followed:

• How Proteins Work (Mike Williamson)

• Introduction to protein structure (Carl Branden &
John Tooze)
• Biochemistry (Lubert Stryer)