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 IP A Control IP Resolve Immune Complex by SDS PAGE WCE Western- Blot with Antibody against B IP A Control IP 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 , 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 : Å, emission ~ 1/d 6 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 of bacteriophage T4 DNA polymerase in vitro. Science 249: (1990). Random oligonucleotide pool Affinity matrix

Yeast One Hybrid Y 1-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 interaction ABA + B AB dissociation rate = k off [AB]association rate = k on [A] [B] At equilibrium:association rate = dissociation rate k on [A] [B] = k off [AB] [A] [B] k off ______ = ___ = K D = dissociation constant (M) [AB] k on [AB] [AB]/[B] [AB] [B]

adrenocorticoid receptor neuropeptide trypsin 8 x Antibody-antigen interaction Lambda rep (monomer/dimer) 2 x lambda rep (dimer/DNA) 1 x Range of Biological Dissociation Constants

Phage Display