1. Discovering Macromolecular Interactions

2. An experimental strategy for identifying new molecular actors in a process candidate approach general screen

3. –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

4. Protein/protein –extracellular –intracellular Protein/nucleic acid Types of Interactions

5. –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

6. 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

7. 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)

8. 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

9. Fluorescence Resonance Energy Transfer: FRET : 10-50 Å, emission ~ 1/d 6 FLIM (Fluorescence lifetime imaging) BiFC (Bimolecular fluorescence complementation)

10. –Electrophoretic mobility shift assay (EMSA) –SELEX –yeast one hybrid –Chromatin immunoprecipitation (ChIP) –Footprinting (in vitro and in vivo) Interaction Methods Protein/DNA

11. Electrophoretic mobility shirt assay (EMSA)

12. SELEX 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). Random oligonucleotide pool Affinity matrix

13. Yeast One Hybrid Y 1-n Bait DNA sequence Library protein TATA Repoter (his, lacZ)

14. Chromatin Immunoprecipitation (ChIP)

15. 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

16. Demonstrate by multiple independent molecular methods ·co-localization ·biochemical affinity/specificity Genetics ·phenotypic overlap between two mutants Verifying a Putative Interaction

17. 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]

18. 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

32. Phage Display