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MN-B-WP II (BInf 2) Bioinformatische Datenbanken

MN-B-WP II (BInf 2) Bioinformatische Datenbanken. Woche 4: Interaktionsdatenbanken. Kay Hofmann – Protein Evolution Group http://www.genetik.uni-koeln.de/groups/Hofmann. Classification of physical protein interactions. By mechanism b inding ( non-covalent ) b inding (covalent )

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MN-B-WP II (BInf 2) Bioinformatische Datenbanken

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

  2. Classificationofphysicalproteininteractions 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 orB ─┤ A By time frame • transient • permanent • covalent) By stoichiometry • binary • multimeric

  3. Binary andcomplexinteractions C A B C A B A B D E E D Several binary interactions can lead to multi-protein complexes A B C A B A B A B C C C 2x binary ternary

  4. Protein complexeswith variable Stoichiometry Kd D A B + D A B C C Equilibrium with sub-complexe D E F A B A B A B A B C C C C Complexwith 'part time' subunits

  5. Example: Proteasome

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

  7. Methodsforestablishingproteininteraction Co-IP, Pulldown YeastTwo-Hybrid Phage Display FRET

  8. Yeasttwo-hybrid system Only if protein X binds to protein Y, the activation domain (AD) will be brought 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

  9. Limits ofthe Y2H method Pro: • Conditionssimilartoin vivo-Situation • Unbiased (proteome-wide) screenspossible Con: • Bothfalse-positive andfalse-negative resultsarecommon • Interaction hastotakeplace in thenucleus • Proteins canshow Y2H interactionthat will nevermeet in real life  Validation withadditional methodmandatory

  10. Co-Immunoprecipitation Protein X with tag A Protein Y mit Tag B Antibodyagainst tag A Antibodyagainst tag B withReporter CellexpressingProtein X CellexpressingProtein Y Celllysis Celllysis Detectionofprotein Y Interaction ofproteinX und Y ? Washingsteps Matrix Matrix Matrix

  11. Pull-down with MS evaluation Analysis Protein X with tag A Celllysis Elution Add proteinmixture Washing Matrix

  12. Limits of Co-IP and Pull-down Pro: • Test proteinscanbeextractedfromthe 'right' cellswith PTMs. • Allowspurificationanddetectionof multi-proteincomplexes Con: • Bothfalse-positive andfalse-negative resultsarecommon • Proteins havetobeextracted in soluble form • Findingthe proper washingandelutionconditionscanbetricky • Indirectinteraction via bridgingfactorsfromthecelllysatecannotbeexcluded  Validation with additional methodmandatory

  13. Interaction databases A large number of protein interaction databases is available, most contain largely overlapping data. Examples : BioGRID, INTACT, MINT Most databases store interactions in binary form, even when derived from complex purifications. There are different strategies for treating complexes. 'hub model' predicts4 binaryinteractions Real Complex Measureddata (A asbait) B C B A A B C C A D E D D E E B C A 'network model' predicts10 binaryinteractions E D

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

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