This document discusses techniques for studying the binding of drugs to proteins using spectroscopy and calorimetry. It first provides background on proteins, their structure and functions. It then discusses protein-drug interactions and experimental techniques like isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism spectroscopy that can be used to characterize binding, including determining affinity, binding sites, and thermodynamic profiles. The techniques provide valuable information for drug design and development by furthering understanding of pharmacokinetics and pharmacodynamics.

1. Binding of drug with Protein using
spectroscopy and calorimetry
techniques
Presented by
Adil Mahammad
GI8956

2. Content :
 What is protein?
 Amino acids
 Functions of proteins
 Structures of protein
i. Primary structure
ii. Secondary structure
iii. Tertiary structure
iv. Quaternary structure
 Protein-drug interaction
 Research plan
 Experimental techniques
i. ITC
ii. Fluorescence spectroscopy
iii. CD spectroscopy
 Application of protein –drug interaction

3. What is protein ?
 The term “protein” was coined by Berzelius in1838.
 Proteins are organic compound with a high molecular weight formed of C, H, O, N and
may also contain S, P , non-protein organic group and metal ions.
 Protein is a large and complex biomolecule consisted of amino acids linked together by
peptide linkage.
 It is a macronutrient that is essential for building muscle mass and regulating all the body
processes.
 They act as biological catalyst ( enzymes), forms structural part of organisms, participate
in different cell reaction.

4. Amino acids :
 Amino acid in the form of protein is the second largest
component of human muscle and other tissues.
 20 Amino acids are naturally incorporated into polypeptides
called proteinogenic or standard amino acids.
 These are divided into two types :
• Essential amino acids: His, Ile, Leu, Met, Phe, Thr, Trp, Val,
Lys.
• Non-essential amino acids: Ala, Arg, Asn, Gln, Gly, Pro, Ser,
Tyr, Cys, Glu, Asp.

5. 20% of human body is made up of proteins which are essential for structure, function, and regulation of the
body tissues and organs.
 They perform various functions such as catalyzing metabolic reactions, DNA replication and transporting
molecules from one location to another.
Functions of Proteins

6. Structures of protein
Primary
structure
Secondary
structure
Tertiary
structure
Quaternary
structure

7.  Primary structure :
 The linear sequence of amino acids in a polypeptide chain.
 Starting from the amino terminal (N) end to the carboxyl terminal (C) end.
 A change of just one amino acid in a protein’s sequence can affect the protein’s overall function
and structure.

8.  Secondary structure:
 H-bond between amino-hydrogen and
carboxyl- oxygen atoms in the peptide
backbone.
 α-Helix: The carbonyl of amino acid 1
form a H-bond to the N-H of amino acid 5.
 β-Sheet: Two or more polypeptide chain
lineup next to each other, forming a sheet
like structure held together by H-bonds.

9. Comparison of α-Helix and β-Sheet
Criteria α-Helix β-Sheet
Structure Coiled rod like Fully extended sheet like
Axial distance between amino
acids
1.5Å 3.5Å
Number of constituent
polypeptide chain
One One or more polypeptide chain
Stabilized by hydrogen bonds
between NH and C=O groups in
The same polypeptide chain Different or same polypeptide
chain
Hydrogen bonds are Parallel to polypeptide
backbone
Perpendicular to polypeptide
backbone

10.  Tertiary structure :
 single polypeptide chain “backbone”
with one or more protein secondary
structures.
 The formation of pockets and sites for
the recognition and binding of ligands.
 Disulfide bonds contribute to tertiary
structure most.

11. Quaternary structure:
 Highest classification level of protein
structure.
 May be composed of two or more smaller
protein chains.
 Multiple folded protein subunits in a multi-
subunit complex.
 E.g : Hemoglobin, DNA polymerase and
Antibodies etc.

12.  Protein-drug Interaction:
 A protein-ligand binding is a spontaneous process which forms a complex of protein bound
with ligand (where ligand means any molecule that binds with protein ).
 It is reversible non-covalent interaction (No sharing of electrons) between two molecules.
 This non-covalent binding may be influenced by these interactions:
 Electrostatic forces
 Van der Waals force
 hydrophobic interactions
 Solvent-related forces
 The binding of drug to plasma proteins is a major determinant of drug disposition.
 Drug-protein binding may be a reversible or an irreversible process.

13. How strong the binding is?
(Affinity)
What is the binding
mechanism?
Where it binds? (Binding site)
How this binding change the
conformation of protein?
What are the thermodynamic
profile for the complex
formation?
Which amino acids and what
type of forces are responsible at
the binding site?
Research plan

14. Experimental Techniques
System: Protein(macromolecule) with Drug(ligand)
Spectroscopy: (a) Fluorescence
(b) Circular dichroism
Calorimetry: Isothermal titration calorimetry

15. Isothermal Titration Calorimetry
15
Fig: Isothermal titration calorimeter (MicroCal ITC200).

16. • Isothermal Titration Calorimetry of the Binding of drug to Protein at Different Temperatures
Schematic diagram of ITC experiment
The binding constant (Kb), binding stoichiometry (n), enthalpy change (∆Ho) and change in
entropy (T∆So),Gibbs free energy (∆Go) have been directly obtained by using ITC
experiment
16

17. Fluorescence spectroscopy
Fig: Fluorescence spectrophotometer (Hitachi-2700).

18. Jablonski diagram of fluorescence

19. Circular Dichroism Spectroscopy
19
Fig: Circular dichroism spectroscopy (JASCO J-815).

20.  CD spectroscopy determinines the structures and monitoring structural changes of biomolecules like
proteins, carbohydrates and nucleic acids.
 Provide valuable information about the secondary structure content like α-helices, β-pleated sheets and β-
turns.

21. Applications of protein-drug interaction :
Protein-drug interaction is an important process in determining the activity, distribution, rate of
excretion, and toxicity of drugs in the body.
It is significant for the treatment of various diseases, providing safer and improved influence.
It is vital to determine therapeutic effect of drugs during drug design and development.
Level of free drug provide valuable information about the dose of drug.
It is very important in understanding the pharmacokinetics and pharmacodynamics of drug which is
useful for pharmaceutical industry.

22. THANK YOU