1. MN-B-WP II (BInf 2) Bioinformatische Datenbanken Kay Hofmann – Protein Evolution Group http://www.genetik.uni-koeln.de/groups/Hofmann Woche 4: Interaktionsdatenbanken

2. By 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

3. Several binary interactions can lead to multi-protein complexes AB ABC D E AB C D E ABCAB C AB C AB C 2x binaryternary

4. AB C D AB C D + KdKd AB C AB C D AB C E AB C F Equilibrium with sub-complexe Complex with 'part time' subunits

6. ub R1 F S1 R2 F S2 R3 F S3 skp1 cullin rbx1 E2 F R SCF is a ubiquitin ligase complex with variable Adaptor-Subunits

7. Methods for establishing protein interaction Yeast Two-Hybrid Co-IP, Pulldown Phage DisplayFRET

8. Only if protein X binds to protein Y, the activation domain (AD) will be brought into proximity of the DNA- binding domaoin (DBD) and start transcription 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

9. 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

10. Co-Immunoprecipitation Cell expressing Protein X Cell expressing Protein Y Cell lysis Matrix Cell lysis Matrix Washing steps Interaction of protein X und Y ? Detection of protein Y Protein X with tag A Protein Y mit Tag B Antibody against tag A Antibody against tag B with Reporter

11. Pull-down with MS evaluation Protein X with tag A Cell lysis Matrix Add protein mixture Washing Elution Analysis

12. Pro: Test proteins can be extracted from the 'right' cells with PTMs. Allows purification and detection of multi-protein complexes Con: 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

13. A large number of protein interaction databases is available, most contain largely overlapping data. Examples : BioGRID, INTACT, MINT ABC D E Most databases store interactions in binary form, even when derived from complex purifications. There are different strategies for treating complexes. Real Complex A B C DE Measured data (A as bait) A B C D E 'hub model' predicts 4 binary interactions A B C D E 'network model' predicts 10 binary interactions

14. A useful method to guess the function of an unknown protein X is to look at functional data of genes/proteins associated with X V V C U U A W W B A physical interactors ? ? co-regulated genes genetic interactors Integrate functional data from different sources