1. 1 Protein-Protein Interactions High-throughput strategy –Prediction from sequence In silico analysis –Protein A from species A: domain 1 and 2 –Protein 1 ’ and protein 2 ’ from species B Recognition  sequence homology –Yeast two-hybrid screen of whole genome –Tagged protein Tandem affinity purification (TAP) + MS Immunoprecipitation + MS –Ab to target proteins –Pooling assay Biochemical functional assay

2. 2 Y2H: basic design If the two proteins don't interact, the reporter gene remains inactive and the cells can't grow on -His plates: If the two proteins interact, the reporter gene (here: HIS3) is switched on and the diploids can grow on -His plates: http://depts.washington.edu./sfields/

3. 3 Discovery Q Y2H protein interaction inside nucleus of yeast cell. Is it OK? What is the proper control? Is it restricted to yeast proteins only?

4. 4 Large-scale Y2H Yeast genome –Array screening Much more time- and labor- intensive More positive identification –Library screening Reasonable time and effort –Bioinfomatics platform http://portal.curagen.com Uetz, et al., Nature 2000 403, 623-627. Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

5. 5 DIP search http://dip.doe-mbi.ucla.edu/ Database of interacting proteins Start/root node 1 st shell nodes 2 nd shell nodes Interactions Color: reliability Width: no. of exp.

6. 6 Protein network Built by association Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

7. 7 Interaction between groups Crosstalk between and within functional groups Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

8. 8 By location Grouped by cellular compartments Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

9. 9 Prediction of functions Guilty-by-association Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. Endocytosis YHR105WYPL246CYGL161C Akr2 Ypt1 Vam7 Yip1 Pep12 Vesicle transport & membrane fusion

10. 10 Tag-protein + MS Co-precipitation –Tandem-affinity purification –SDS-PAGE Mass spectrometry Bioinformatics Kumar and Snyder, 2002 Nature 415, 123-124

11. 11 Pooling functional assay Biochemical assay for activity –6144 GST-ORF strains –64 pools of 96 fusions/plate each –Pools of 12 columns and 8 columns 12 8 1 1

12. Priori and Potentials Priori –The GST-ORF is functional –Soluble after extraction –Remain functional Retains other required components when purified Fast and sensitive Potentials –Determine the range of the substrate proteins –Identifying gene leads to the binding of particular molecule, ligand, or drug.

13. 13 Next Week Please read Text p. 183-187. –1:30 pm at room A105 –Advanced handout? Paper discussion –Whatever we did not finish today Homework assignment –How far did you get?