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Protein-protein interactions. Masoud Youssefi, MD,PhD Division of microbiology/virology. Introduction. important field in cell biology, biochemistry Localization and trafficking posttranslational modifications signaling networks also important field in viral replication

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protein protein interactions

Protein-protein interactions

Masoud Youssefi, MD,PhD

Division of microbiology/virology

introduction
Introduction
  • important field in cell biology, biochemistry

Localization and trafficking

posttranslational modifications

signaling networks

  • also important field in viral replication
  • very difficult to predict
  • two main patterns:

■ domain-domain interactions

■domain-peptide interactions

an example virion assembly
An example: virion assembly
  • The components come together and the Nucleocapsid is formed which in turn will become completed to the whole particle.
  • The assembly process begins when concentration of structural proteins is enough within the cell to drive the process.
  • Many protein-protein, protein-nucleic acid and in case of membrane viruses protein-membrane (fatty acid) interactions are needed.
the mechanism of interaction
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
overview of techniques
Overview of techniques
  • Gel filtration
  • Far western blot
  • Affinity chromatography
  • Co-immunopercipitation
  • Capillary elecrophoresis
  • Biosensor
  • FRET microscopy
  • Confocal microscopy
  • 2 hybrid assay
  • Protein microarry
  • Maspec
  • NMR
  • Co-crystallization for crystallography
gel filtration chromatography
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
far western blot
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
co immunoprecipitation
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
affinity chromatography
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
fret cont
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

yeast two hybrid assay1
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!
mamalian two hybrid assay
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
what are biosensors
what are biosensors?
  • Transducer converts physical change(heat, change in charge, light absorbance, mass) into an electrical signal
confocal microscopy
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)
overview of techniques1
Overview of techniques
  • Gel filtration
  • Far western blot
  • Affinity chromatography
  • Co-immunopercipitation
  • Capillary elecrophoresis
  • FRET microscopy
  • Confocal microscopy
  • 2 hybrid assay
  • Maspec
  • NMR
  • Co-crystallization for crystallography