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

B

HIS3DBD

BORF AD ORF AD

HIS3DBD

B ADORF

HIS3DBD

B

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

Analysis of Yeast Protein Complexes by Mass spec

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

Analysis of Yeast Protein Complexes by Mass spec

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

Eft1

Hrt1

Rav1

Rav2

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

Skp1

A

B

C

D

E

F

G

H

I

JK

L

M

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

4

log-odds score:

log2 (-------)

observed frequencyof pair

frequenciesof domains

Pij

PiPj

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