Pt.

Ravishankar Shukla university 1

Raipur (c.g.)
Biophysical presentation

Topic – ligand protein binding

Session – 2025-26
Guided By :
presented by :
Mr. pushpkant sahu shriya
chaturvedi
riya
patel
 Contents 2

1. Protein ligand binding kinetics

2. Thermodynamic relationship
3. Thermodynamic parameters
4. Methods of studying protein ligand binding
5. factors affecting protein ligand interaction
6. Applications of protein ligand binding
 Protein ligand binding kinetics
3
Protein ligand binding kinetics :
Protein ligand binding kinetics describes the rate at which the ligand and protein bind to
each other.
P + L PL
Where,
PL = Protein ligand complex
Kon – Rate constant for forward binding
Koff - Rate constant for reverse unbinding
The units of Kon and Koff – M−1·s−1 and s−1
At equilibrium , the forward reaction should be balanced by reverse unbinding reaction :
Kon [P][L] = Koff [PL]
The binding constant Kb (in unit of 1 / M) is defined by :
Kb = = =
Where , Kd (unit of M) is called dissociation constant.
 Thermodynamic relationship
4
• Protein ligand system is the thermodynamic system in which a very
complex interaction occur with heat exchange.

• Gibb’s free energy measure the capacity of thermodynamic system

to do maximum or reversible work at constant temperature and
pressure . The driving forces characterised by the Gibb’s free energy
which are responsible for association of protein and ligand.

• Spontaneity of reaction : Protein ligand binding occur only when the

change in Gibb’s free energy of the system is negative , when the
system is in equilibrium state at constant temperature and constant
pressure.

• The protein ligand association extent is determine by the magnitude

of negative ∆G, it determines the stability of protein ligand
complex .
 Relationship between Standard Gibbs
5
free energy and Binding constant :
• The binding affinity of a ligand to a protein is quantified by the binding
constant (Kb).
• The relationship between ΔG° and Kb is given by:
• ΔG°= − RTlnKb​
where:
• ΔG° = Standard Gibbs free energy change (kJ/mol)
• R = Universal gas constant (8.314 J/mol·K)
• T = Temperature in Kelvin
• kb = binding constant
Interpretation:
• ΔG° < 0 → Spontaneous binding (favourable interaction)
• More negative ΔG° → Higher Kb→ Stronger binding
• Less negative ΔG° → Lower K → Weaker binding
 Thermodynamic parameters
6

Gibbs Free Energy Equation:

ΔG=ΔH−TΔS
Where:
• ΔG (Gibbs Free Energy): Determines whether
binding is spontaneous.
• ΔH (Enthalpy Change): Heat released or
absorbed during binding.
• T (Temperature): Affects entropy-driven
interactions.
• ΔS (Entropy Change): Degree of disorder in
the system.
 Bound fraction
 7
In practice , we often determine the fraction of active sites occupied by the ligand
and the total number of active binding site present in the protein . The fraction of
occupied binding site (Ѳ).
Kb =
 Substituting [PL] with ka[L][P]
 Eliminating [P] and rearrangement gives the result in terms of equilibrium
association constant.
Ѳ=
Ѳ=
Ѳ=
 Ѳ=
Ѳ=

8
Fraction of bound sites depends upon the concentration of ligand and K d.
• Hill Equation : 9

• n is the Hill coefficient, which reflects the degree of cooperativity:

• N = 1 , non-cooperative binding
• N > 1 > , positive cooperativity
• N <1 n , negative cooperativity
 Methods for studying Protein ligand 10
binding
1. Isothermal Titration Calorimetry (ITC)
• Measures heat changes during binding.
• Provides binding affinity (Kₐ), enthalpy (ΔH), and entropy (ΔS).
2. Surface Plasmon Resonance (SPR)
• Monitors real-time binding without labelling.
• Measures binding kinetics (association/dissociation rates, Kₐ, Kd).
3. Fluorescence Spectroscopy
• Uses ligand or protein fluorescence changes.
• Provides binding constants (Kₐ) and conformational changes.
 Factors affecting protein ligand
11
binding

1. Intrinsic Factors (Molecular Properties)

• Protein Conformation & Flexibility
• Binding sites must be properly structured for ligand recognition.
• Induced fit vs. lock-and-key mechanism.
• Ligand Size, Shape, and Functional Groups
• Small ligands may bind more easily, but larger ligands may offer stronger
interactions.
• Functional groups determine hydrogen bonding, electrostatic, and hydrophobic
interactions.
 12
Molecular Interactions
🔹 Hydrogen bonding (stabilizes binding)
🔹 Van der Waals forces (weak but cumulative)
🔹 Electrostatic interactions (charged residues attract ligands)
🔹 Hydrophobic effects (nonpolar regions enhance binding)
2. Temperature & pH
Temperature: Too high can denature proteins, too low slows binding.
pH: Affects ionization of amino acids and ligand structure.
3. Protein Conformational Flexibility
Some proteins undergo structural changes (induced fit mechanism).
Rigid proteins may limit ligand access.
.
 13
4. Ligand Structure & Modifications
• Shape, size, and functional groups influence binding affinity.
Modifications (e.g., adding functional groups) can enhance interaction.
5. Binding Site Properties
 Active site accessibility and polarity affect ligand entry.
Presence of cofactors or competing molecules may interfere.
 Applications of protein
14
ligand binding
1. Drug Discovery & Pharmacology
💊 Target Identification: Finding drug-binding sites on
proteins.
🔬 Drug Design: Developing inhibitors/activators for
diseases (e.g., COVID-19 antivirals).
🧪 Structure-Based Drug Design (SBDD): Using
molecular docking to design better drugs.
2. Enzyme Regulation & Biocatalysis
 ⚙️Competitive & Non-Competitive Inhibition:
Controls enzyme activity in metabolic pathways.
🧫 Biotechnological Enzyme Engineering: Designing
enzymes with improved efficiency.
 15
3. Signal Transduction & Cellular
Communication
📡 Hormone-Receptor Binding: Regulating
physiological processes (e.g., insulin binding to
its receptor).
🧬 Neurotransmitter Binding: Essential for
brain function and synaptic signaling.
4. Biosensors & Diagnostics
🩸 Biosensors for Disease Detection: Using
protein-ligand interactions to detect biomolecules
(e.g., glucose sensors).
🔬 Antibody-Antigen Binding in ELISA Tests:
Used for diagnosing infections (e.g., COVID-19,
HIV).
 16
5. Protein-Ligand Binding in
Structural Biology
• X-ray Crystallography & NMR
Spectroscopy: Studying protein-ligand
complexes for drug development.
Molecular Dynamics Simulations:
Predicting ligand binding in virtual
screening.
References - 17

 https://pmc.ncbi.nlm.nih.gov/articles/PMC4783878/
 You tube channel : fundamentals of biochemistry
 Gray matter
 https://www.nature.com/articles/s41598-024-72784-3