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

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

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

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

DIP search

http://dip.doe-mbi.ucla.edu/

• Database of interacting proteins

Start/root node

1st shell nodes

2nd shell nodes

InteractionsColor: reliabilityWidth: no. of exp.

6

Protein network• Built by

association

Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

7

Interaction between groups

• Crosstalk between and within functional groups

Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

8

By location• Grouped by cellular compartments

Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

9

Prediction of functions• Guilty-by-association

Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.

Endocytosis

YHR105WYPL246C YGL161C

Akr2Ypt1

Vam7

Yip1

Pep12

Vesicle transport & membrane fusion

10

Tag-protein + MS

• Co-precipitation– Tandem-affinity

purification– SDS-PAGE

• Mass spectrometry

• Bioinformatics

Kumar and Snyder, 2002 Nature 415, 123-124

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

11

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

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?