Protein-Protein Interactions (PPIs)

PPIs: Types, Significance

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

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

7. X-Ray Crystallography output

8. 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.

9. 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.

10. 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.

11. 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.

12. 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

13. 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

14. 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.

15. 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.

16. 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

17. 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.

18. 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