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Proteins by Salman Ul Islam.
This document discusses methods for detecting protein-protein interactions. It begins by introducing the importance of protein-protein interactions for cell biology and biochemistry. It then describes several common techniques for studying these interactions, including gel filtration chromatography, co-immunoprecipitation, affinity chromatography, fluorescence resonance energy transfer (FRET) microscopy, yeast two-hybrid assays, and confocal microscopy. The document emphasizes that protein-protein interactions are non-covalent and reversible, and detecting them requires accurate techniques.
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Proteins by Salman Ul Islam.
- 1. Detecting Protein-Protein interactions Salman UlIslam (MS) Cellular Bio Chemistry Lab
- 2. CONTENTS Introduction Types of protein-proteininteractions Methods of detection
- 3. INTRODUCTION Importance for cellbiology 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 Nitrogenouscompounds, 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 •
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- 6. FORMATION OF ADIPEPTIDE
- 7. THE MECHANISM OFINTERACTION Non-covalent so reversible Van del waals forces Hydrophobic interactions Electrostatic bonds Hydrogen bonds For strong couplings very accurate force field potentials are needed
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- 9. WHY ARE PROTEIN-PROTEININTERACTIONS 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 OTHERFIELDS • 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 OFPROTEIN INTERACTIONS • Binary protein-protein interactions • Scaffolding proteins
- 12. THE TYPES OFPROTEIN 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
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- 14. HOW TO STUDYPROTEIN PROTEIN INTERACTION?
- 15. OVERVIEW OF TECHNIQUES • • • • • • Gelfiltration 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 Alsocalled ”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 Alsocalled ”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 bindsto 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 thecase 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
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- 21. FRET CONT Cyan fluorescenceprotein (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 HYBRIDASSAY 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!
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- 25. MAMALIAN TWO-HYBRID ASSAY Isanalogous 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 goodtechnique to detect intracellular co-localization of proteins Point scan laser system minimizes overlaps in image (perfect for imaging Co-localization of proteins)
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