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

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

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.

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

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

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

X-Ray Crystallography

output

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.

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.

• 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.

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.

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

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

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.

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.

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

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.

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