Download LOCALIZATION OF A MOLECULE

Discovering Macromolecular
Interactions
An experimental strategy for
identifying new molecular actors in
a process
candidate approach
general screen
Some situations in which this
strategy could be applied
– 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
Types of Interactions
Protein/protein
– extracellular
– intracellular
Protein/nucleic acid
Interaction Methods
– 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
Co-Immunoprecipitation
Control IP WCE
IP
A
A
Control IP WCE
IP
A
B
IP protein A
Resolve Immune
Complex by SDS PAGE
WesternBlot with
Antibody against B
Tandem Affinity Purification (TAP)
Advantages
- Specificity
- good for complex
- PTM/localization
Drawbacks
-need verification
-not quantitative
-not as sensitive as 2 hyb (for transient)
SILAC
(Stable Isotope Labeling of Amino-Acid in Cell Culture)
Yeast Two Hybrid
DNA binding domain hybrid
Advantages
-sensitivity
Activation domain encoded by a library
Drawbacks
-lack of specificity
-False positives
-problems with PTM
-problems with localization
Interaction
Gal1-lacZ (blue colonies)
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
Fluorescence Resonance
Energy Transfer: FRET
FLIM
(Fluorescence lifetime imaging)
BiFC
(Bimolecular fluorescence
complementation)
: 10-50 Å, emission ~ 1/d6
Interaction Methods Protein/DNA
–
–
–
–
–
Electrophoretic mobility shift assay (EMSA)
SELEX
yeast one hybrid
Chromatin immunoprecipitation (ChIP)
Footprinting (in vitro and in vivo)
Electrophoretic mobility shirt
assay (EMSA)
SELEX
Random oligonucleotide
pool
Affinity
matrix
elute
clone & sequence
C.Tuerk, L. Gold Systematic evolution of high-affinity RNA ligands of
bacteriophage T4 DNA polymerase in vitro. Science 249:505-510 (1990).
Yeast One Hybrid
Y1-n
Library protein
TATA
Bait DNA sequence
Repoter (his, lacZ)
Chromatin Immunoprecipitation
(ChIP)
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
Verifying a Putative Interaction
Demonstrate by multiple independent
molecular methods
·co-localization
·biochemical affinity/specificity
Genetics
·phenotypic overlap between two mutants
Equilibrium constant measures
the strength of interaction
A+B
AB
association rate = kon [A] [B]
AB
A+B
dissociation rate = koff [AB]
At equilibrium: association rate = dissociation rate
kon [A] [B] = koff [AB]
[AB]
[AB]/[B]
[A] [B]
koff
______ = ___ = KD = dissociation constant (M)
[AB]
kon
[B]
[AB]
Range of Biological Dissociation
Constants
•
•
•
•
•
•
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
Phage Display