The document discusses protein-protein interactions (PPIs), including an introduction to PPIs, the types of interactions, techniques used to study them like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy, and factors that affect PPIs. It also covers methods to investigate PPIs such as affinity purification coupled with mass spectrometry and yeast two-hybrid screening. Applications of understanding PPIs include developing therapeutic drugs and identifying functions of unknown proteins.

1. Topic: Protein-Protein Interactions
Presented by: D.Sairam
Course: Bioorganic and Bioinorganic
Chemistry
Course Code: BSBT-211
Course Instructor: Dr. Vineet Sharma
Presentation Code: U3P1

2. Overview of the Presentation
Introduction
Types of Protein- Protein Interactions
Techniques used to study them
Factors affecting these Interactions
Methods used to Investigate Interactions
Applications of Protein- Protein Interactions

3. Introduction
• Proteins are the workhorses that facilitate most biological processes
in a cell, including gene expression, cell growth, proliferation,
nutrient uptake, morphology, motility, intercellular communication
and apoptosis.
• Protein–protein interactions (PPIs) refer to intentional physical
contacts established between two or more proteins as a result of
biochemical events and/or electrostatic forces.
• These interactions are very important in our lives as any disorder in
them can lead to fatal diseases such as Alzheimer’s and Creutzfeld-
Jacob Disease.
• Perhaps the most well known example of Protein-Protein
Interaction is between Actin and Myosin while regulating Muscular
contraction in our body.
• Another prominent application of PPIs is in Signal Transmission
inside the lining of a cell or along the boundaries of various cell.

4. Types of Protein-Protein Interactions
On the basis of their Composition
 Homo-Oligomers: These are macromolecule complexes having one type
of protein subunits.
E.g. : PPIs in Muscle Contraction
 Hetero-Oligomers: These are macromolecule complexes having multiple
types protein subunits.
E.g. : PPI between Cytochrome Oxidase and TRPC3 (Transient receptor
potential cation channels

5. On the basis of their Duration of Interaction
• Stable Interactions: These comprise of interactions that last for a
long duration. These Interactions carry out Functional or Structural
roles.
E.g.: Haemoglobin structure
• Transient Interactions : Interactions that last a short period of
time.
E.g.: Muscle Contraction

7. Techniques Used to study structure of Protein Complexes
There are three major techniques used to study the structure of Protein
Complexes.
1. X-Ray Crystallography
2. Protein NMR Spectroscopy
3. Cryo microscopy

8. X-Ray Crystallography
output

9. X –Ray Crystallography
• Devised by Sir John Cowdery Kendrew, X-ray Crystallography
is the oldest of the three methods used for examining Protein
structures.
• He solved the structure of Myoglobin in Whale.
• This technique uses beams of X-rays that are diffracted by
crystals on a film and thus, producing the image of the proteins
on the film.
• It reveals a 3-Dimensional view of the protein.

10. Protein NMR Spectroscopy
• It is a field of structural biology in which NMR spectroscopy is used to
obtain information about the structure and dynamics of proteins, and
also nucleic acids, and their complexes.
• The field was pioneered by Richard R. Ernst and Kurt Wüthrich who
were awarded the Nobel for their contributions in 2002.
• Structure determination by NMR spectroscopy usually consists of
several phases, each using a separate set of highly specialized
techniques.
• The sample is prepared, measurements are made, interpretive
approaches are applied, and a structure is calculated and validated.

11. • The blue arrows represent
the orientation of the N – H
bond of selected peptide
bonds.
• Protein structure is
calculated by determining
orientation of the bond and
influence of Magnetic
Field.

12. Cryo Electron Microscopy
• This is a form of transmission electron microscopy (EM) where the
sample is studied at cryogenic temperatures (generally liquid
nitrogen temperatures).
• This technique was devised by a group of scientists led by Frank Joachim
in EMBL i.e. European Molecular Biology Laboratory.
• Their biggest advantage is that the specimen does not need to be stained
unlike the other two methods.
• However, the resolution of the molecule obtained is not as good as the
other two techniques.
• This technique is still very much a work in progress technique.

13. Factors affecting PPIs
• Protein concentration, which in turn are affected by expression levels and
degradation rates
• Protein affinity for proteins or other binding ligands;
• Ligands concentrations (substrates, ions, etc.);
• Presence of other proteins, nucleic acids, and ions;
• Electric fields around proteins.
• Occurrence of covalent modifications

14. Methods to Investigate PPIs
• There are many methods to investigate Protein-Protein Interactions namely
o-immuno-precipitation, protein microarrays, analytical
ultracentrifugation, light scattering, fluorescence spectroscopy, Resonance-
energy transfer systems, Surface Plasmon resonance, protein-fragment
complementation assay, and Calorimetry etc…
• The two most prominent methods used for investigating PPIs are:
• Affinity purification coupled to mass spectrometry
• Yeast two-hybrid screening

15. Yeast two-hybrid screening Technique
 It was first proven using Saccharomyces cerevisiae as biological model by
Fields and Song.
 Yeast cells are transfected with two plasmids: Prey and Bait
 Bait comprises of protein of interest with DNA binding domain factor like
GaI4.
 Prey has cDNA portion that can active the above domain.
 Thus, transcription does not take place until both of them bind.

16. Disadvantages of Yeast two hybrid screening technique
• It’s specificity is very low.
• Number of PPIs identified are low as they are lost during
purification.
• As it uses Yeast as a model organism, it can have trouble while
studying other organisms.

17. Affinity purification coupled to mass spectrometry
• Affinity purification coupled to mass spectrometry mostly
detects stable interactions and thus is better than Yeast 2 hybrid
method.
• This method starts by purification of the tagged protein.
• One of the most advantageous and widely used method to
purify proteins with very low contaminating background is
the Tandem affinity purification
• This was developed by Bertrand Seraphin and Mathias Mann

18. Applications of PPIs
 Many PPIs are being used as therapeutic targets as they exhibit properties such
as allosteric sites.
 Maraviroc, a drug that inhibits CCR5 gp 120 interaction and is a prominent
anti HIV drug.
 Recently , a group of scientists were able to develop 30 peptides using
protein–protein interaction studies to inhibit telomerase recruitment towards
telomeres.
 PPIs have been used to identify the functions of unknown proteins.
 It is based on the assumption that uncharacterized proteins have similar
functions as their interacting proteins
 YbeB, a protein of unknown function was found to interact with ribosomal
proteins and later shown to be involved in translation.

19. References
• http://www.pnas.org/content/106/16/6706.full.pdf
• http://www.trpchannel.org/interactions/show?trp=TRPC3&interactor=
Cytochrome+c+oxidase+5A&type=Interactor
• https://dx.doi.org/10.1016%2Fj.ymeth.2012.07.015
• https://dx.doi.org/10.1093%2Fbioinformatics%2Fbti514
• https://dx.doi.org/10.1016%2Fj.tips.2013.04.007
• http://www.quintarabio.com/screen
• http://bioinfo3d.cs.tau.ac.il/MAPPIS/mappis_info.html
• http://www.wisegeek.com/what-are-purkinje-cells.htm
• http://www.trpchannel.org/proteins/show?id=Cytochrome+c+oxidase+
5A
• http://www.piercenet.com/method/overview-protein-protein-
interaction-analysis