Detecting
Protein-Protein interactions

Salman Ul Islam (MS)
Cellular Bio Chemistry Lab
CONTENTS


Introduction



Types of protein-protein interactions



Methods of detection
INTRODUCTION


Importance for cell biology and biochemistry
Localization and trafficking
posttranslational modifications
...
CHARACTERISTICS OF PROTEINS
Nitrogenous compounds, contain carbon, hydrogen,
oxygen, nitrogen, and sulfur
• Basic building...
AMINO ACID
FORMATION OF A DIPEPTIDE
THE MECHANISM OF INTERACTION
Non-covalent so reversible
 Van del waals forces
 Hydrophobic interactions
 Electrostatic ...
POLYPEPTIDE CHAIN STRUCTURE
WHY ARE PROTEIN-PROTEIN INTERACTIONS SO
IMPORTANT?
The binding of one signaling protein to another can have a
number of co...
IMPACT ON OTHER FIELDS
• Cancer Biology
The study of protein-protein interactions has provided
important insights into the...
THE TYPES OF PROTEIN INTERACTIONS

• Binary protein-protein
interactions
• Scaffolding proteins
THE TYPES OF PROTEIN INTERACTIONS
-ANOTHER CLASSIFICATION

• Metabolic and signaling (genetic)pathways
• Morphogenic pathw...
MORPHOGENIC PATHWAYS
HOW TO STUDY PROTEIN PROTEIN
INTERACTION?
OVERVIEW OF TECHNIQUES
•

•
•

•

•

•

Gel filtration
Far western blot
Affinity
chromatography
Coimmunopercipitation
Capi...
GEL FILTRATION CHROMATOGRAPHY








Also called ”Size
exclusion”
Porous made up of
cross-linked polymers
Small molec...
FAR WESTERN BLOT








Also called ”Blot overlay”
Fractionating proteins on
SDS-PAGE
Blotting to nitocellulose or
...
CO-IMMUNOPRECIPITATION









Protein A binds to
antibodies
Sepharose beads coated
with protein A
Specific antibod...
AFFINITY CHROMATOGRAPHY


In the case of His- tagged proteins



The His-tagged protein binds to nickel or cobalt column...
FLUORESCENCE RESONANCE ENERGY
TRANSFER (FRET)
FRET CONT







Cyan fluorescence protein (CFP) and yellow fluorescence
protein (YFP) are spectral variants of GFP
P...
FRET USING CFP & YFP
YEAST TWO HYBRID ASSAY








Transcription factor, Gal4p, has
DNA binding (BD)(aa1-147) and
transcriptional
activato...
2 HYBRID SYSTEM
MAMALIAN TWO-HYBRID ASSAY






Is analogous to Y2H assay
Plasmids: 1)Gal4pBD-fusion vector
2)VP16AD-fusion vector(vir...
WHAT ARE BIOSENSORS?

• Transducer converts physical change(heat, change
in charge, light absorbance, mass) into an
electr...
CONFOCAL MICROSCOPY



A good technique to detect intracellular co-localization of
proteins



Point scan laser system m...
CONFOCAL MICROSCOPY CONT.
Thanks
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Proteins by Salman Ul Islam.

  1. 1. Detecting Protein-Protein interactions Salman Ul Islam (MS) Cellular Bio Chemistry Lab
  2. 2. CONTENTS  Introduction  Types of protein-protein interactions  Methods of detection
  3. 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. 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. 5. AMINO ACID
  6. 6. FORMATION OF A DIPEPTIDE
  7. 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. 8. POLYPEPTIDE CHAIN STRUCTURE
  9. 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. 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. 11. THE TYPES OF PROTEIN INTERACTIONS • Binary protein-protein interactions • Scaffolding proteins
  12. 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. 13. MORPHOGENIC PATHWAYS
  14. 14. HOW TO STUDY PROTEIN PROTEIN INTERACTION?
  15. 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. 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. 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. 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. 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. 20. FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
  21. 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. 22. FRET USING CFP & YFP
  23. 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. 24. 2 HYBRID SYSTEM
  25. 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. 26. WHAT ARE BIOSENSORS? • Transducer converts physical change(heat, change in charge, light absorbance, mass) into an electrical signal
  27. 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. 28. CONFOCAL MICROSCOPY CONT.
  29. 29. Thanks

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