Post on 27-Dec-2015
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MN-B-WP II (BInf 2) Bioinformatische Datenbanken
Kay Hofmann – Protein Evolution Grouphttp://www.genetik.uni-koeln.de/groups/Hofmann
Woche 4: Interaktionsdatenbanken
Classification of physical protein interactionsBy mechanism· binding (non-covalent)· binding (covalent)· Modification (e.g. phosphorylation)· Cleavage, degradation· Folding (e.g. chaperones)
By effect· undirected· Activating A → B or B → A· Inhibiting A ─┤B or B ─┤ A
By time frame· transient· permanent· covalent)
By stoichiometry· binary· multimeric
Binary and complex interactions
Several binary interactions can lead to multi-protein complexes
A B A B C
DE
A B
C
DE
A B C A B
C
A B
C
A B
C
2x binary ternary
Protein complexes with variable Stoichiometry
A BC
DA B
C
D+
Kd
A BC A B
C
D
A BC
E
A BC
F
Equilibrium with sub-complexe
Complex with 'part time' subunits
Example: SCF-complexes
ub
R1
F
S1
R2
F
S2
R3
F
S3
skp1
cullin
rbx1
E2F
R SCF is a ubiquitin ligase complex with variable Adaptor-Subunits
Yeast two-hybrid system
Only if protein X binds to protein Y,the activation domain (AD) will bebrought into proximity of the DNA-binding domaoin (DBD) and starttranscription of the reporter gene.
Typically, Y2H will be performed in yeast cells that require the reporter gene for viability. In a proteome-wide screen, the surviving cells can be picked and the identity of the Y protein is determined
Pro:• Conditions similar to in vivo-Situation• Unbiased (proteome-wide) screens possible
Con:• Both false-positive and false-negative results are common• Interaction has to take place in the nucleus• Proteins can show Y2H interaction that will never meet in real
life
Limits of the Y2H method
Validation with additional method mandatory
Co-Immunoprecipitation
Cell expressing Protein X
Cell expressing Protein Y
Cell lysis
Mat
rix
Cell lysisM
atrix
Matrix
Washing steps
Interaction of protein X und Y ?
Detection of protein Y
Protein X with tag A
Protein Y mit Tag B
Antibody againsttag AAntibody againsttag B with Reporter
Pull-down with MS evaluation
Protein X with tag A
Cell lysis
Mat
rix
Add proteinmixture Washing
Elution
Analysis
Pro:• Test proteins can be extracted from the 'right' cells with PTMs.• Allows purification and detection of multi-protein complexesCon:• Both false-positive and false-negative results are common• Proteins have to be extracted in soluble form• Finding the proper washing and elution conditions can be
tricky• Indirect interaction via bridging factors from the cell lysate
cannot be excluded
Limits of Co-IP and Pull-down
Validation with additional method mandatory
Interaction databases
A large number of protein interaction databases is available, most contain largely overlapping data. Examples : BioGRID, INTACT, MINT
A B C
DE
Most databases store interactions in binary form, even when derived from complex purifications. There are different strategies for treating complexes.
Real Complex
AB C
D E
Measured data (A as bait)
A
B C
D E
'hub model' predicts4 binary interactions
A
B C
D E
'network model' predicts 10 binary interactions