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Protein-Protein Interactions (PPIs)

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Protein protein interaction
Protein protein interaction
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Protein-Protein Interactions (PPIs)

  1. 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. 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. 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. 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. 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. 6. 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
  7. 7. X-Ray Crystallography output
  8. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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

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