Proteins by Salman Ul Islam.

  • 242 views
Uploaded on

 

More in: Technology
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
  • God job, keep it up, my one suggestion to you that if the slides background were white then it too good.
    Are you sure you want to
    Your message goes here
    Be the first to like this
No Downloads

Views

Total Views
242
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
13
Comments
1
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

  • 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