Protein interactions: main methods for detection

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Protein interactions: main methods for detection (all organisms)

Two-hybrid 8,446(Co-)Immunoprecipitation 567Interaction adhesion assay 225In vitro binding 138Affinity Column 120Copurification 85Immunoblotting 83Other Biophysical 73

All interactions 10,432

Source: The Database of Interacting Proteins (DIP)

Protein interactions: top 8 organisms

Yeast (Saccharomyces cerevisiae)7,881Helicobacter pylori 1,418Man (Homo sapiens ) 595Worm (Caenorhabditis elegans) ~500Mouse (Mus musculus ) 130Fly (Drosophila melanogaster) 81Escherichia coli 56Rat (Rattus norvegicus ) 48

All interactions 10,432

Source: The Database of Interacting Proteins (DIP)

Up to 6,200 Protein interactions reported in 5 papers:

• Fromont-Racine et al.1997, 2000 ~300• Uetz et al./Drees et al. 2000, 2001 ~1,200• Ito et al. 2000, 2001 841/~4,700

==========~2,500 - 6,200

• Mass spectrometry• Protein chips (arrays)• Biochemical proteomics

Extented by recent contributions from

Up to 6,200 Protein interactions reported in 5 papers:

• Fromont-Racine et al.1997, 2000 ~300• Uetz et al./Drees et al. 2000, 2001 ~1,200• Ito et al. 2000, 2001 841/~4,700

==========~2,500 - 6,200

Two-hybrid-Array (rich media)

HIS3DBD

B ADORF

HIS3DBD

BORF AD ORF AD

HIS3DBD

B ADORF

HIS3DBD

BORF AD ORF AD

Two-hybrid-Array (-His media) bait: PCF11

Details in Cagney & Uetz, Methods Enzymol. 328: 3 (2000); Current Protocols in Protein Science 19.6 (2001)

rich - Histidine

Insert movie 1 here Insert movie 2 here

Analysis of Yeast Protein Complexes by Mass spec

Gavin et al. (2002) Nature 415: 141-147

1739 epitope-tagged proteins (include. 1143 human orthologues)1167 clones expressed the tagged protein589 successful purifications232 distinct complexes identified134 new complexes

16,830 protein bands identified (corresponding to 1440 proteins)

Note: The probability to detect the same protein in a complex twice is 70%

Conclusion: there are about 700 complexes in yeast

How many protein interactions are there based on mass spec data?

On average 12 proteins per complex,

Minimum: 11 interactions Maximum: 55 interactions

8,00038,500 interactions total

Our two-hybrid array (!) screens for comparison

~650 baits screened against whole-genome array330 baits yielded ~1200 reproducible interactions

of those 1200 only ~100 showed up in complexes

76 proteins used as both baits/“entry points” by Gavin et al. yielded 227 interactions in our two-hybrid screens (i.e. 2.9 on average)

76 purifications = complexes would contain ~900 proteins with x interactions

Cdc53Cep3

Rcy1 (F)

YBR280 (F)

YJL149 (F)YMR258C (F)

MS1Seol et al. 2001Nature CellBiol. 3: 384

MS3 (Aebersold et al., pers. comm.)

Grr1 (F)

YLR097C (F)

Bdf1Cdc4

Ctf13 (F)Met30 (F)

Rub1Sgt1

YLR224W (F)

YLR352W (F)

YLR368W (F)

Two-HybridUetz et al. 2000Nature 403: 623

protein pulled downwith epitope-taggedprotein B

protein pulled downwith epitope-taggedprotein E

two-hybridinteractions

Two-hybrid…vs. mass spec

Limitations of current network graphs

- static- no localization data

- no or limited integration with other compounds- no or limited data on experimental conditions- no or limited data on expression levels

Predicting Protein Interaction Domains

Based on Sprinzak & Margalit, J. Mol. Biol. 311: 681 [2001]

Protein 1 Protein 2

Protein pairs

Domain information from the Interpro database (Apweiler et al., Nucl. Acids Res. 29; 37 [2001])

Conclusion:

Predicting Protein Interaction Domains

Based on Sprinzak & Margalit, J. Mol. Biol. 311: 681 [2001]

domain combinations

log-odds score:

log2 (-------)

observed frequencyof pair

frequenciesof domains

Summary (sort of…)

Protein interactions in the Database of Interacting Proteins (DIP)

All interactions 10,432PDB entries ~1900one domain mapped 252both domains mapped 175Dissociation constant available26

Source: The Database of Interacting Proteins (DIP) as of late 2001

Protein interactions: main methods for detection

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