INTERACTIONS DEFINES STRUCTURES OF PROTEIN

MPS-301 (E) :
STRUCTURES,
PROPERTIES, AND
FUNCTIONS OF
BIOMOLECULES

BY JITESH BARMAN

DEPARTMENT OF PHYSICS, BHU DATE – 06-11-2023

 Tertiary structure of proteins
➢ Protein tertiary structure is the three dimensional shape of a protein,
consisting only one polypeptide chain.
➢ Protein tertiary structure is very The Structure of
important from the structural and Myoglobin
functional point of view
➢ Tertiary structure play a decisive role
for
• Binging sites of ligands (protein-
drug interactions)
• Active sites of an enzymes
• Binding sites for other proteins
(protein-protein interactions)
➢ Spatial folding of any protein is
required to understand , in addition to
the former biological phenomena, how
the particular mutations or variations
in the gene that encodes a particular
protein lead to changes in the
protein’s behavior.
 Protein folding mechanism
o fff

Process Biochemistry 51 (2016) 1183–1192

 Anfinsen’s dogma or thermodynamic hypothesis
o The hypothesis states that the three dimensional native structure of protein in its
normal physiological environment is the one in which the Gibbs free energy is the
lowest and its conformation is determined by the amino acid sequence.

o Experiments carried out by Christian Anfinsen in the

1950s

o Purified ribonuclease is completely denatured when

concentrated urea solution (8M) containing reducing
agent (2-mercaptoethanol) is added and results in
inactive protein

o Removing the urea and reducing agent completely

restored the enzymatic activity of ribonuclease as it
refolded into the correct tertiary structure

o This was the first evidence that all the information

required for correct folding of a protein is contained
in its amino acid sequence
 Protein folding pathways : thermodynamics point of view
o Protein folding is a highly complex process,
and several models have been proposed
o In one model, protein folding is seen as a
hierarchical process
o First local secondary structures are formed
o followed by formation of stable super
secondary structures
o In an alternative model, hydrophobic forces
among the nonpolar residues initiate the
folding by a spontaneous collapse of the
polypeptide into a compact state (molten
globule) having high content of secondary
structures.
o The process of protein folding can be seen as
a folding funnel
o The unfolded state (top of the funnel) has a
higher degree of entropy and free energy
o Progress of folding is accompanied by a
decrease in the number of states present,
thereby reducing the entropy and free energy
and increasing the amounts of native like
structures of the protein
Introduction to Biomolecular Structure and Biophysics, by G. Mishra
 Folding/unfolding of protein
o Proteins undergo unfolding in aqueous solution upon addition of certain reagents
(denaturing agents)
o Guanidine hydrochloride or urea in high concentrations
o Denaturation with these chemicals is one of the primary ways of measuring the
conformational stability of proteins and comparing the stabilities of mutant proteins
o The exact nature of the molecular interaction of denaturant molecules with protein
surfaces is not well understood
o It is known that the interaction between the urea and GnHCl molecule and the protein
is more favourable than the interaction among the groups of the proteins in water
o These denaturation process alters the equilibrium between the native (folded) and
denatured (unfolded )
o ⇌
o The free energy of the unfolding reaction at a particular denaturant concentration is

o Δ =− ln ;
o Where and are the concentration of protein in the folded or native
conformation, and unfolded or denatured conformations respectively
o The relative concentrations of and can be measured easily by spectroscopic
methods
o The equation only works for a system with two states ; folded and unfolded
𝑈
𝐺
𝑅
𝑇
𝐹𝑈𝐹𝑈
𝐹
𝑈
𝐹
 Concentration of denaturant (m) value
o The free energy of unfolding is plotted against denaturant concentration in the
transition region
o Their interaction with proteins shows a linear dependence of free energy of unfolding
on the molar concentration of denaturant
o Using linear extrapolation method (LEM) two parameters were obtained: the free
H2O
energy of unfolding at zero denaturant concentration (represented by, Δ , the
intercept) and the dependence of free energy on denaturant concentration
(represented by the symbol m, the slope)

Protein Science (1995), 4:2138-2148

𝐺
 Tertiary structure of proteins
➢ The unique folding of a protein is the result of non-bonded interactions
between polypeptide backbone and the side chains, acting co-operatively
➢ These non-bonded interactions which stabilise the proper folding of the
macromolecules
• Ionic (Salt birdge) and dipole-dipole
• Van der Waals and hydrophobic interactions
• Hydrogen bonds
 Ionic (salt bridge) in macromolecules
➢ Madelung equation for binding energy
2
=− + (− ) where and are empirical parameters
𝑖
𝑗
𝑟
𝑖
𝑗
𝑈
𝜆
𝑒
𝜌
𝜆𝜌
𝑞
𝑖
𝑗
𝑟
 Dipole-dipole interactions
➢ van der Waals interactions are dipole-dipole, dipole-induced dipole and
London interactions
➢ Average energy of interaction of permanent dipoles can induce dipole
moments by Keesom equation
2 2
2 1 2 1
=−
3 6
𝜀
𝑟
𝐾
𝑇
𝑈
𝜇
𝜇
 Dipole-induced dipole interactions
➢ Induced dipole moment =
➢ Energy of interaction is given by the Debye equation
2
2
=− 6
𝜀
𝑟
𝑈
𝑖
𝑛
𝑑
𝜇
𝛼
𝐸
𝛼
𝜇
 Induced dipole-induced dipole interactions
2
3
➢ London equation =− , where is the first ionization potential
4 6
➢ Lennard-Jones Potential
=− 6
+ 12
𝑟
𝑟
𝑟
𝑈
𝑈
𝐼
𝐼
𝛼
𝐴
𝐵
 In next class
➢ Hydrophobic interactions, water and its properties, hydration shell

➢ The Quaternary structure of proteins

➢ Protein classes

➢ Fibrous protein, Globular proteins and other classes of proteins

Protein folding and forces responsible for it
Hydrogen bond
 Hydrogen bond and properties of water
➢ Hydrogen bonds are not unique to water; they exist to varying degrees
between electronegative atoms (e.g., O, N, F, and Cl) and H atoms
covalently bound to similar electronegative atoms
➢ These bonds are special in that they only involve hydrogen atoms, which,
by virtue of their tendency to become positively polarized and their
uniquely small size, can interact strongly with nearby electronegative
atoms, resulting in an effective H-mediated “bond” between two
electronegative atoms
➢ Water and ice shows cage kind of structure due to Hydrogen bonding
 Hydration shell of water

➢ In water, each protein is surrounded by a hydration shell of water

molecules which directly interact with protein molecules
➢ Multiple H-bonds are formed between the protein and the water molecules
➢ Water –mediated transformations cause disruptions in the protein bound
water molecules (first hydration shell) and these disturbances in turn
manifest in structural perturbances
➢ These perturbances appear to be more pronounced at the ligand binding
regions of protein and enzymes
 Characteristics of amino acid side chain in H-bond
➢ Hydrogen bond donors only
 Characteristics of amino acid side chain in H-bond
➢ Hydrogen bond donors and acceptors
 Characteristics of amino acid side chain in H-bond
➢ Hydrogen bond donors and aceptors
Hydrophobic effect
 Hydrophobic effect and Hydrophobic interactions

➢ Hydrophobic interactions are non-directional, weak but of high importance

in protein folding and stability of the higher order structure
➢ The formation of non-polar interaction occurs through hydrophobic
interactions
➢ Hydrophobicity depends on surface area and dipole content
Summary of the forces in macromolecules
 The quaternary structure
➢ The structural conformations with a single polypeptide chain is completed
at tertiary level. Examples are ; myoglobins, lysozymes, cytochrome c,
trypsin and chymotrypsin etc.
➢ Protein structure with two or more polypeptide chains are called
quaternary structure
➢ Hemolobins, cytochrome oxidase are examples of quaternary structure.
➢ Non-polar interactions, which are weak, non-directional and driven by
entropy factors, play a major role in the structural organization and stability
of quaternary structure
➢ Minor and subtle alterations in the interactions between subunits (subunits
associations) in a multimeric protein complex lead to large range structural
changes
Hemoglobin
 Protein classes
➢ From structural point of view : globular and fibrous molecules
➢ Globular proteins have both single polypeptide chains and enzymes
(myoglobins, lysozymes, cytochrome c etc.) and multi-subunit proteins and
complexes (hemoglobins. ATPases etc.)
➢ Globular proteins have compact globular shapes due to chain reversals of
polypeptide backbones and packing of side and are amenable to
crystallization in a single-crystalline form
➢ Fibrous proteins and protein complexes (viruses), whose axial lengths are
far greater than their widths, are semicrystalline
➢ Periodic along the fibre axis
 Fibrous proteins
➢ Fibrous proteins groups
• The collagen group
• The keratin (k-m-e-f) group
• Others
➢ The collagen group:
✓ Found in skin, tendon, blood vessels, bone and teeth
✓ The structural unit is tropocollagen
• Rod shaped
• Length ~ 3000 Å and width ~ 15 Å
• Molecular mass ~ 285 kilodaltons
• Consists of three polypeptide chains
• Amino acid compositions: glycine ~ 33 %, proline ~ 15 – 30 %,
and hydroxyproline (Hypr) ~ 15 to 30 %
• Sequence is Gly-X-Y and Y- is generally 4 – hydroxyproline
• Three left hended helices are wound each other forming a
coiled coil right handed triple helix, a cable like structure
 The collagen group
✓ Trans hydrogen bonds between neighboring chains residues, proper
docking of proline and hydroxyproline residues and other stabilizing
interactions such as hydroxylation and covalent cross-links by lysine side
chains render structural strength to collagen fibres
✓ Lack of proper packing, and defective hydroxylation or cross-links, lead to
molecular diseases like scurvy, bone and skindeformities
 In next class
➢ Keratin (k-m-e-f) group proteins

➢ Other classes

➢ Globular proteins

▪ Motifs

▪ Domains

▪ Other types of structural motifs

 Thank you

Namaskaar !!