Discovering Macromolecular Interactions

1. Discovering Macromolecular Interactions

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

3. Some situations in which this strategy could be applied • receptors or ligands without partners • intracellular signaling molecules • regulatory sequence with unknown transcription factor • transcription factor with unknown target • search for a partner of a transcription factor • telltale motif such as SH3, RING, coiled coil

4. Some Types of Interactions • protein/protein • extracellular • disulphide bonds and glycosylation • intracellular • protein/nucleic acid

5. Interaction Methods • co-immunoprecipitation • glutathione-S-transferase (GST) pull down • surface plasmon resonance (BiaCore) • FRET: 10-50 Å, emmission ~ 1/d6 • chemical cross-linking • yeast two hybrid • co-purification • chromatography, centrifugation, tandem affinity purification (TAP) • solution binding- Scatchard analysis

6. Interaction Methods continued • gel overlay • phage display/expression libraries • Electrophoretic mobility shift assay (EMSA) • SELEX • yeast one hybrid • Chromatin immunoprecipitation (ChIP) • footprinting • filter binding

7. Yeast Two Hybrid Controls? 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

8. Tandem Affinity Purification (TAP)

9. Yeast One Hybrid Y1-n

10. Methods to Identify Gene Targets of a Transcription Factor? • expression profiling combined with genomic sequence analysis • ChIP on a chip • SELEX combined with sequence analysis • genetics combined with other methods

11. elute clone & sequence SELEX C.Tuerk, L. Gold Systematic evolution of high-affinity RNA ligands of bacteriophage T4 DNA polymerase in vitro. Science 249:505-510 (1990).

12. Chromatin Immunoprecipitation (ChIP)

13. ChIP on a chip afterR. Young et al.

14. Verifying a Putative Interaction • demonstrate by multiple independent molecular methods • co-localization • biochemical affinity/specificity • genetics • phenotypic overlap between two mutants • what is the best genetic evidence of direct interaction?

15. Equilibrium Constant Measures Strength of Interaction A + B AB AB A + B association rate = kon [A] [B] dissociation rate = koff [AB] At equilibrium: association rate = dissociation rate kon [A] [B] = koff [AB] [A] [B] koff ______= ___ = KD = dissociation constant (M) [AB] kon

16. Range of Biological Dissociation Constants QUAL ALERT! • adrenocorticoid receptor 10-10 • neuropeptide 10-9 • trypsin 8 x 10-5 • Ab/Ag interaction 10-5 - 10-12 • Lambda rep (monomer/dimer) 2 x 10-8 • lambda rep (dimer/DNA) 1 x 10-10 Concentration of one molecule per typical mammalian cell volume is about 10-12 M.

17. Other ways of achieving specificity of interaction • avidity (multivalent interaction) • subcellular compartmentalization • restricted expression • activation or cofactors