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Proteins by Salman Ul Islam.

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  • 1. Detecting Protein-Protein interactions Salman Ul Islam (MS) Cellular Bio Chemistry Lab
  • 2. CONTENTS  Introduction  Types of protein-protein interactions  Methods of detection
  • 3. INTRODUCTION  Importance for cell biology and biochemistry Localization and trafficking posttranslational modifications signaling networks  Essential in viral replication Difficult to predict two main patterns: domain-domain interactions domain-peptide interactions  
  • 4. CHARACTERISTICS OF PROTEINS Nitrogenous compounds, contain carbon, hydrogen, oxygen, nitrogen, and sulfur • Basic building block is the amino acid • Serve as structural components of animals • Serve as control molecules (enzymes) • Serve as transport and messenger molecules •
  • 5. AMINO ACID
  • 6. FORMATION OF A DIPEPTIDE
  • 7. THE MECHANISM OF INTERACTION Non-covalent so reversible  Van del waals forces  Hydrophobic interactions  Electrostatic bonds  Hydrogen bonds  For strong couplings very accurate force field potentials are needed 
  • 8. POLYPEPTIDE CHAIN STRUCTURE
  • 9. WHY ARE PROTEIN-PROTEIN INTERACTIONS SO IMPORTANT? The binding of one signaling protein to another can have a number of consequences: • Such binding can serve to recruit a signaling protein to a location where it is activated and/or where it is needed to carry out its function. • The binding of one protein to another can induce conformational changes that affect activity or accessibility of additional binding domains, permitting additional protein interactions.
  • 10. IMPACT ON OTHER FIELDS • Cancer Biology The study of protein-protein interactions has provided important insights into the functions of many of the known oncogenes, tumor suppressors, and DNA repair proteins. • Pharmacogenetics Pharmacogenetic research has expanded to include the study of drug transporters, drug receptors, and drug targets.
  • 11. THE TYPES OF PROTEIN INTERACTIONS • Binary protein-protein interactions • Scaffolding proteins
  • 12. THE TYPES OF PROTEIN INTERACTIONS -ANOTHER CLASSIFICATION • Metabolic and signaling (genetic)pathways • Morphogenic pathways in which groups of proteins participate in the same cellular function during a developmental process • Structural complexes and molecular machines in which numerous macromolecules are brought together
  • 13. MORPHOGENIC PATHWAYS
  • 14. HOW TO STUDY PROTEIN PROTEIN INTERACTION?
  • 15. OVERVIEW OF TECHNIQUES • • • • • • Gel filtration Far western blot Affinity chromatography Coimmunopercipitation Capillary electrophoresis Biosensor • • • • • • • FRET microscopy Confocal microscopy 2 hybrid assay Protein microarry Maspec NMR Co-crystallization for crystallography
  • 16. GEL FILTRATION CHROMATOGRAPHY     Also called ”Size exclusion” Porous made up of cross-linked polymers Small molecules are trapped by the beads For self assembling proteins monomers come later
  • 17. FAR WESTERN BLOT       Also called ”Blot overlay” Fractionating proteins on SDS-PAGE Blotting to nitocellulose or PVDF membrane Overlaying with a solution of the protein of interest Binding the added protein to an immobilized protein on the membrane Detection with antibody against the overlaying protein
  • 18. CO-IMMUNOPRECIPITATION       Protein A binds to antibodies Sepharose beads coated with protein A Specific antibody binds to the protein of interest The complex is precipitated by binding to the beads via protein A Proteins are released from beads by boiling Western blot
  • 19. AFFINITY CHROMATOGRAPHY  In the case of His- tagged proteins  The His-tagged protein binds to nickel or cobalt column  His-tagged protein and it’s associated protein are eluted from the column by adding imidazole
  • 20. FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
  • 21. FRET CONT      Cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP) are spectral variants of GFP Plasmid constructs to fuse the proteins of interest to CFP and YFP Co-transfection of plasmids to the cells Fixation of the cells and view by confocal microscopy Disadvantage:False negative results: If the fluorophores are over 200Ǻ apart while the proteins interact with each other, no signal will be observed
  • 22. FRET USING CFP & YFP
  • 23. YEAST TWO HYBRID ASSAY     Transcription factor, Gal4p, has DNA binding (BD)(aa1-147) and transcriptional activator(AD)(aa768-881) domains Stimulates transcription at a promoter reconized by Gal4p (upstream activating sequence,UAS) Lac Z reporter gene encodes beta-galactosidase which produces blue pigment when the colony is grown in a media containing X-Gal Disadvantage: time consuming!
  • 24. 2 HYBRID SYSTEM
  • 25. MAMALIAN TWO-HYBRID ASSAY     Is analogous to Y2H assay Plasmids: 1)Gal4pBD-fusion vector 2)VP16AD-fusion vector(viral activator) 3)luciferase reporter plasmid contaning multiple copies of Gal4p binding sites(UAS) Co-transfection: in the case of interaction, luciferase activity will be detected Advantage: good for studying mammalian proteins: they may not fold correctly in yeast or they may require post-tranlational modifications for protein interaction
  • 26. WHAT ARE BIOSENSORS? • Transducer converts physical change(heat, change in charge, light absorbance, mass) into an electrical signal
  • 27. CONFOCAL MICROSCOPY  A good technique to detect intracellular co-localization of proteins  Point scan laser system minimizes overlaps in image (perfect for imaging Co-localization of proteins)
  • 28. CONFOCAL MICROSCOPY CONT.
  • 29. Thanks