discovering macromolecular interactions. an experimental strategy for identifying new molecular...
TRANSCRIPT
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Discovering Macromolecular Interactions
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An experimental strategy for identifying new molecular actors in a process
candidate approach
general screen
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– receptors or ligands without partners
– intracellular molecules (enzyme/substrate)
– Motifs such as SH2, SH3, RING, coiled coil
– regulatory sequence with unknown transcription factor
– transcription factor with unknown target gene
Some situations in which this strategy could be
applied
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Protein/protein– extracellular
– intracellular
Protein/nucleic acid
Types of Interactions
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– co-immunoprecipitation– glutathione-S-transferase (GST) pull down
– co-purification– chromatography, tandem affinity purification (TAP)
– yeast two hybrid– phage display/expression libraries– FRET– solution binding- Scatchard analysis
Interaction Methods
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Co-Immunoprecipitation
A B
IP protein A
IPA
ControlIP
Resolve ImmuneComplex by SDS PAGE
WCE
Western-Blot withAntibody against B
IPA
ControlIP
WCE
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Tandem Affinity Purification (TAP)
SILAC(Stable Isotope Labeling of Amino-Acid in Cell Culture)
Advantages- Specificity- good for complex- PTM/localization
Drawbacks-need verification-not quantitative-not as sensitive as 2 hyb (for transient)
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Yeast Two Hybrid
CHIEN, CT, BARTEL, PL, STERNGLANZ, R, AND FlELDS, S The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9578-9582, November 1991
Gal1-lacZ (blue colonies)
Activation domain encoded by a library
DNA binding domain hybrid
Interaction
Advantages-sensitivity
Drawbacks-lack of specificity-False positives-problems with PTM-problems with localization
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Fluorescence Resonance Energy Transfer: FRET
: 10-50 Å, emission ~ 1/d6
FLIM (Fluorescence lifetime imaging)
BiFC(Bimolecular fluorescence
complementation)
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– Electrophoretic mobility shift assay (EMSA)
– SELEX– yeast one hybrid– Chromatin immunoprecipitation (ChIP)– Footprinting (in vitro and in vivo)
Interaction Methods Protein/DNA
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Electrophoretic mobility shirt assay (EMSA)
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SELEX
elute
clone & sequence
C.Tuerk, L. Gold Systematic evolution of high-affinity RNA ligands ofbacteriophage T4 DNA polymerase in vitro. Science 249:505-510 (1990).
Random oligonucleotidepool
Affinitymatrix
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Yeast One Hybrid
Y1-n
Bait DNA sequence
Library protein
TATA
Repoter (his, lacZ)
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Chromatin Immunoprecipitation (ChIP)
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Methods to Identify Gene Targets of a Transcription
Factor?•expression profiling combined with genomic sequence analysis
•ChIP followed by UHTS
•SELEX combined with sequence analysis
•genetics combined with other methods
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Demonstrate by multiple independent molecular methods·co-localization·biochemical affinity/specificity
Genetics·phenotypic overlap between two mutants
Verifying a Putative Interaction
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Equilibrium constant measures the strength of
interactionAB A + BA + B AB
dissociation rate = koff [AB] association rate = kon [A] [B]
At equilibrium: association rate = dissociation rate
kon [A] [B] = koff [AB]
[A] [B] koff
______ = ___ = KD = dissociation constant (M) [AB] kon
[AB
]
[AB
]/[B
]
[AB][B]
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• adrenocorticoid receptor 10-10
• neuropeptide 10-9
• trypsin 8 x 10-5
• Antibody-antigen interaction 10-5 - 10-12
• Lambda rep (monomer/dimer) 2 x 10-8
• lambda rep (dimer/DNA) 1 x 10-10
Range of Biological Dissociation Constants
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Phage Display