BSE 102 Bioscience

Spring 2024
Proteins
Instructor: Dr. Sunil Kumar Boda
Assistant Professor
Department of Biosciences & Biomedical Engineering
IIT Indore
E-mail: sunilboda@iiti.ac.in
 Molecules of life - Proteins
Diverse biological roles of proteins:
• Biological catalysts – enzymes (trypsin, pepsin, alkaline phosphatase, lysozyme)
• Hormones/ signaling proteins – Insulin, glucagon
• Transport proteins embedded in cell membranes – help move sugar and other nutrients into cells. Eg: Transthyretin,
a transport protein in blood helps in vitamin A and thyroid hormone transport throughout the body
• Structural proteins – extracellular matrix proteins (collagen, elastin, keratin, fibroin)
- cell cytoskeleton (actin, tubulin, intermediate filaments)
• Defense against pathogens – antibodies

Proteins – polymers made up of monomers called amino acids. Amino acids are linked by peptide or amide bonds and
form polypeptides

All proteins are constructed from a ubiquitous set of 20 amino acids. Cells can produce proteins with strikingly different
activities and properties by joining the same set of 20 amino acids in different combinations and sequences.

Light produced by firefly enzyme luciferase Erythrocytes contain oxygen-transporting Protein keratin is the chief structural component
catalysis of protein luciferin with ATP hemoglobin of horns, hair, horns, scales, feathers
 Common structural features of amino acids

α-carbon atom is a chiral center, when R ≠ H

All naturally occurring amino-acids are L-amino acids
 Peptide bond formation UV absorbance characteristics of proteins

Amino acids with

aromatic R groups for
absorbance at 280 nm – a
characteristic feature of
protein
Peptide/ amide bond is formed between two amino
acids by removal of H2O. By convention, sequence is
written from N-terminus to C-terminus.

Disulfide bond formation

 Amino acids can act as weak acids and bases - zwitterions
The characteristic pH at which the net electric charge on an amino acid/ protein is zero is called Isoelectric pH or Isoelectric
point, designated as pI.

Isoelectric pH

For Glycine with R = H (non-ionizable side chain),

1 1
pI = 2(pK1 + pK2) = 2(2.34 + 9.60) = 5.97
 Titration curve for (b) histidine
Problem: Estimate the pI of the dipeptide (Ala-Lys) given:
 Levels of structures in proteins
What makes a protein an enzyme or a hormone or a structural protein or an antibody? The most obvious differences are in
the protein structure, that occur at different levels.
• Primary structure – sequence of amino acids
• Secondary structure – stable arrangement of amino acid residues giving rise to recurring structural patterns
• Tertiary structure – 3D folding of individual polypeptide subunit
• Quaternary structure – arrangement of 2 or more polypeptide subunits in space
 Primary structure - Determining amino acid sequence of a protein
Traditional protein sequencing: α-amino group of the amino acid residue Mass Spectrometry
at the amino-terminus of the polypeptide is labelled with FDNB, dansyl
or dabsyl chloride

Sanger’s method

Edman degradation

Phenylthiocarbamoyl
 Secondary structure of proteins
Secondary structure – local structure of the polypeptide stabilized by intramolecular and intermolecular
hydrogen bonding. Common secondary protein structures include α – helix and β sheet.

α-helix: polypeptide backbone is wound around an imaginary longitudinal axis with the amino acid R
groups extending outward.
Repeating unit is a single turn of helix which extends 5.4 Å along the longitudinal axis. Each helical turn
includes 3.6 amino acid residues.
Optimal use of internal hydrogen bonding confers stability to α-helical conformation.
Alanine stabilizes α-helix while glycine and proline destabilize the helix.
Right handed α-helix is common. Eg: α-keratin, myoglobin, hemoglobin

β strands – self assemble into β sheets and stabilized by inter-strand hydrogen bonding
β sheet - parallel and antiparallel. Eg: silk fibroin, keratin
Backbone of the polypeptide chain extended into a zigzag structure. Arrangement of several β
conformation segments side by side leads to β-sheet structure.
R groups of adjacent amino acids protrude from the zigzag structure in opposite directions creating an
alternating pattern.
Hydrogen bonds form between adjacent segments of the polypeptide chain within the sheet.
Interstrand H-bonds are in-line in antiparallel β-sheet , while they are distorted or not-in-line in the
parallel variant.
Adjacent polypeptide chains in a β-sheet can be parallel or antiparallel (having same or opposite amino-
to-carboxyl orientations, respectively).
 Torsion angles in peptides/ proteins: Ramachandran Plot
Peptide bond is planar and rigid Three dihedral/ torsion angles in polypeptide chain:
Phi (φ) – involves the C—N—Cα—C bonds and rotation about N-Cα
Psi (ψ) – involves the N—Cα—C—N bonds and rotation about C-Cα
Omega (ω) – peptide bond; trans ± 180 °
Both φ and ψ are defined as ± 180 ° when the polypeptide is fully
extended and all peptide groups are in the same plane.
Dihedral angles in Omega (ω) = 180 ° (trans) is sterically more favorable
H2O2 Omega (ω) = 0 ° (Cis) is sterically less favorable
Ramachandran Plot: Plot of torsional angles (angles between two
planes) - Psi (ψ) vs Phi (φ) of amino acids contained in a peptide.
Plot shows the range of permissible angles and the types of
structure that can be adopted by polypeptides.

Ramachandran plot : between two dihedral angles ψ and φ.

G.N. Ramachandran
ψ and φ can take any value between -180° and +180°
(1922-2001)
Allowed values of ψ and φ are dictated by the steric hinderance
between atoms in the polypeptide chain resulting from the amino acid
side chain groups. Eg: For smaller side chains like Glycine, R = H, greater
degree of rotation is possible leading to larger ψ and φ values. For larger
side chains like Proline, degree of rotation about C-Cα and N-Cα is
sterically limited, thus leading to smaller ψ and φ values
A conformation in which both ψ and φ are 0° is not allowed.
 Protein – Tertiary and Quaternary Structure
Tertiary structure – overall three dimensional arrangement of all atoms in a protein

While secondary structure refers to spatial arrangement of adjacent amino acid residues in a
segment of the polypeptide, tertiary structure involves long range interaction of amino acids
that are far apart in the polypeptide sequence and in different types of secondary structures
leading to completely folded protein or its subunit. Eg: Myoglobin is composed of 8 α-helices.

Weak non-covalent interactions and sometimes covalent disulfide links hold the interacting Myoglobin
segments in their characteristic tertiary positions.

Quaternary structure – For proteins with two or more polypeptide chains or subunits, the
arrangement of the subunits in three dimensional complexes constitutes quaternary
structure. Eg: Hemoglobin is composed of 4 sub-units: 2 β-globin and 2 α-globin

Based on higher order structures, two classes of proteins –

Hemoglobin
Fibrous proteins: polypeptide chains arranged in long strands or sheets
Globular proteins: polypeptide chains folded into spherical or globular shape
 Fibrous proteins are adapted for structural function
Fibrous proteins – insoluble in water due to high concentration of hydrophobic amino
acid residues in the interior and outer surface of the protein. Eg: Collagen, α-keratin, silk
fibroin, etc.

Fibrous proteins give strength and flexibility to structures they occur in.

α-Keratin – belong to the family of intermediate filament (IF) proteins.

α-Keratin is a right-handed α-helix. Pairs of α-Keratin are wound to form two-chain coils.
These are further organized into higher order structures called protofilaments and
protofibrils. About four protofibrils – 32 strands of α-Keratin are combine to form an
intermediate filament.

Strength of fibrous proteins is a result of covalent crosslinks between the polypeptide

chains. In α-Keratins, the crosslinks stabilizing the quaternary structure are disulfide
bonds.

Hair straightening – based on reduction, curling and oxidizing disulfide bonds in α-keratin

In hardest and toughest α-Keratins like rhinoceros horns, up to 18% of the residues are
cysteines involved in disulfide bonds.
 Globular proteins – Mb and Hb
Globular proteins – segments of polypeptide chains fold more compactly than seen in
fibrous proteins. Globular proteins include enzymes, transport proteins, motor
proteins, regulatory proteins and immunoglobulins.

Myoglobin – relatively small (Mr 16,700), oxygen binding protein of the muscles. It
functions to store oxygen and diffuse oxygen in rapidly contracting muscle tissue.

Myoglobin – comprised of a single polypeptide chain of 153 amino acids and a single
protoporphyrin or heme group unlike hemoglobin with 4 heme groups. Both are deep
red/brown in color due to the heme groups.

The flat heme groups rests in a crevice or pocket in the myoglobin molecule. Iron (Fe2+)
has two types of coordination: 4 in a plane bonded to flat porphyrin and 2
perpendicular with one bonded to ‘N’ from a histidine residue and other for binding to
O2.

Hemoglobin (Mr 64,500) composed of four polypeptide chains in the globular protein
part (two identical α chains of 141 residues + two identical β chains of 146 residues)
and four heme groups with iron as Fe2+. Hb can be described as a tetramer of 2α+2β.
 Hydrophobicity dictates differential folding in globular and membrane proteins
Residue Globular protein Membrane protein
Non-polar residues (V, L, I, M, F, Present in the interior of the Present on the surface due to
Y, W) protein as a hydrophobic core hydrophobic interaction with
membrane lipid bilayer
Polar charged residues (R, K, D, Present on protein surface as Present in the hydrophilic core
E, H) catalytic sites of the protein that provide
channels for ion transport (Na+
and K+)
Polar neutral residues (S, T, N, Q, Hydrogen bond network Inside surface – part of the ion
Y, W) channel

--- ---
Hydrophilic residues

Hydrophobic residues
--- ---
 Protein misfolding and disease

Diseases associated with protein misfolding – type 2 diabetes, Alzheimer, Huntington and
Parkinson’s disease. Collectively called Amyloidoses.

Misfolding mechanism – a soluble protein secreted by the cells in the misfolded state is
converted into an insoluble extracellular amyloid fiber. Fibers are highly ordered, unbranched,
7-10 nm diameter and a high degree of β-sheet structure. The β-sheet segments are oriented
perpendicular to the axis of the fiber. In some amyloid fibers, overall structure features two
layers of β-sheet, such as that shown for amyloid-β-peptide.

Proteins that form amyloid fibrils have a higher concentration of aromatic amino acids in the
core of the β-sheet or α-helix. The proteins secreted in an incompletely folded conformation,
eg: two β-sheets from incompletely folded protein molecules fold to form amyloid fibrils.
Outside of the growing fibril with the β-sheet core, the outside proteins can fold differently.

Normally, proteins undergo partial folding in the endoplasmic reticulum (ER). Under stress or
overwhelming protein synthesis exceeding their folding rate in the ER, unfolded proteins
accumulate. As an unfolded protein response (UPR), transcriptional regulators increase
chaperone concentrations or activate proteases and autophagy.