Post on 04-Jan-2016
description
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