1 Protein-Protein Interactions High-throughput strategy –Prediction from sequence In silico...
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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
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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/
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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?
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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.
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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.
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Protein network• Built by
association
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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Interaction between groups
• Crosstalk between and within functional groups
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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By location• Grouped by cellular compartments
Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261.
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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
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Tag-protein + MS
• Co-precipitation– Tandem-affinity
purification– SDS-PAGE
• Mass spectrometry
• Bioinformatics
Kumar and Snyder, 2002 Nature 415, 123-124
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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
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8
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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.
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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?