protein-protein interactions (ppis)
TRANSCRIPT
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
• 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