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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)
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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)
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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
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• 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
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Two-hybrid-Array (rich media)
HIS3DBD
B ADORF
HIS3DBD
B
HIS3DBD
BORF AD ORF AD
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HIS3DBD
B ADORF
HIS3DBD
B
HIS3DBD
BORF AD ORF AD
Two-hybrid-Array (-His media) bait: PCF11
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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
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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
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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
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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
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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
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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
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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
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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:
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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
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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