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
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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
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
15. OVERVIEW OF TECHNIQUES
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Gel filtration
Far western blot
Affinity
chromatography
Coimmunopercipitation
Capillary
electrophoresis
Biosensor
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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
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
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!
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)
