Discovering Macromolecular Interactions

An experimental strategy for identifying new molecular actors in a process

candidate approach

general screen

– 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

Protein/protein– extracellular

– intracellular

Protein/nucleic acid

Types of Interactions

– 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

Co-Immunoprecipitation

A B

IP protein A

IPA

ControlIP

Resolve ImmuneComplex by SDS PAGE

WCE

Western-Blot withAntibody against B

IPA

ControlIP

WCE

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)

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

Fluorescence Resonance Energy Transfer: FRET

: 10-50 Å, emission ~ 1/d6

FLIM (Fluorescence lifetime imaging)

BiFC(Bimolecular fluorescence

complementation)

– Electrophoretic mobility shift assay (EMSA)

– SELEX– yeast one hybrid– Chromatin immunoprecipitation (ChIP)– Footprinting (in vitro and in vivo)

Interaction Methods Protein/DNA

Electrophoretic mobility shirt assay (EMSA)

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

Yeast One Hybrid

Y1-n

Bait DNA sequence

Library protein

TATA

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

Demonstrate by multiple independent molecular methods·co-localization·biochemical affinity/specificity

Genetics·phenotypic overlap between two mutants

Verifying a Putative Interaction

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]

• 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

Phage Display