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9. Vorlesung WS 2004/05Softwarewerkzeuge1 V9: Protein-Protein-Wechselwirkung - Consurf - Russell & Aloy Server - Webseite mit Proteindomänen - Beispiel.

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Presentation on theme: "9. Vorlesung WS 2004/05Softwarewerkzeuge1 V9: Protein-Protein-Wechselwirkung - Consurf - Russell & Aloy Server - Webseite mit Proteindomänen - Beispiel."— Presentation transcript:

1 9. Vorlesung WS 2004/05Softwarewerkzeuge1 V9: Protein-Protein-Wechselwirkung - Consurf - Russell & Aloy Server - Webseite mit Proteindomänen - Beispiel zu Protein-Protein Wechselwirkung Cytochrom c: Cytochrom c Oxidase Barnase:Barstar - Klausurvorbereitung

2 9. Vorlesung WS 2004/05Softwarewerkzeuge2 Consurf - degree of conservation at each amino acid site is inversely related to its rate of evolution. -aim: identify functional regions on protein surface by mapping degree of sequence conservation within protein family -Use phylogenetic trees instead of MSA: by a weighting scheme reduce influence of redundant sequences

3 9. Vorlesung WS 2004/05Softwarewerkzeuge3 InterPreTS Protein-protein interactions are structurally conserved for > 30% sequence identity. Predict complexes of A:B based on sequence homology to A and B when structure of A:B complex is available.

4 9. Vorlesung WS 2004/05Softwarewerkzeuge4 V9: Protein-Protein-Wechselwirkung Pawson Lab

5 9. Vorlesung WS 2004/05Softwarewerkzeuge5 SH2 domains – universally used protein family Pawson Lab

6 9. Vorlesung WS 2004/05Softwarewerkzeuge6 Proteins using SH2 domains Pawson Lab

7 9. Vorlesung WS 2004/05Softwarewerkzeuge7 Binding properties of SH2 domains Pawson Lab

8 9. Vorlesung WS 2004/05Softwarewerkzeuge8 Binding properties of SH2 domains Pawson Lab

9 9. Vorlesung WS 2004/05Softwarewerkzeuge9 WW domain /research.html Pawson Lab

10 9. Vorlesung WS 2004/05Softwarewerkzeuge domain /research.html Pawson Lab

11 9. Vorlesung WS 2004/05Softwarewerkzeuge domain /research.html Pawson Lab

12 9. Vorlesung WS 2004/05Softwarewerkzeuge domain /research.html Pawson Lab

13 9. Vorlesung WS 2004/05Softwarewerkzeuge13 Introduction: Photosynthesis

14 9. Vorlesung WS 2004/05Softwarewerkzeuge14 Docking strategy No X-ray structure available for complex cyt c552:COX docking. Flöck, Helms (2002) Proteins 47, 75

15 9. Vorlesung WS 2004/05Softwarewerkzeuge15 Protein-protein docking of cyt c552 and COX Exercise on model system: Complex of yeast Cytochrome c Peroxidase with iso-1-Cytochrome c X-ray structure (Kraut et al. 1992) Heme positions of crystal complex and 19 best docked and energy-minimized complexes. Crystal complex has lowest energy. Docked complex with second-best energy has RMSD of only 2.0 Å. Flöck, Helms (2002) Proteins 47, 75

16 9. Vorlesung WS 2004/05Softwarewerkzeuge16 Protein-protein docking of cyt c552 and COX Superposition of complexes of Cyt c / Cyt c552 with COX (bovine) COX (P.d.) Additonal loop of bovine COX collides with c552 (grey). Flöck, Helms (2002) Proteins 47, 75

17 9. Vorlesung WS 2004/05Softwarewerkzeuge17 Protein-protein docking of horse heart cyt c and COX Best predicted complex of horse heart cytochrome c with cytochrome c oxidase from Paracoccus denitrificans. Almost identical with best structure of Roberts et al. for complex of horse heart cytochrome c with bovine cytochrome c oxidase. Docking with DOT-program (1999). Flöck, Helms (2002) Proteins 47, 75

18 9. Vorlesung WS 2004/05Softwarewerkzeuge18 Two favorable docking positions Two favorable docking positions for cyt c552 with COX. The conformation of the flexible linker and of the N-terminal helical anchor are fictitous. Flöck, Helms (2002) Proteins 47, 75

19 9. Vorlesung WS 2004/05Softwarewerkzeuge19 kinetic on-rates of protein-protein complexes from Brownian Dynamics simulations McCammon group website (UCSD)

20 9. Vorlesung WS 2004/05Softwarewerkzeuge20 kinetic on-rates: exp data Mutationk on 10 6 [M -1 s -1 ] horse heart cyt c : COX Wild type oxidase3.7 D135N0.3 N160D2.8 Ionic strength [mM] P.d. cyt c 552 : COX refs:Drousou, Malatesta, Ludwig, Eur J Biochem (2002) 269, 2980 Maneg, Ludwig, Malatesta, J Biol Chem (2003) 278, 46734

21 9. Vorlesung WS 2004/05Softwarewerkzeuge21 brownian dynamics details Electrostatics computed with programs UHBD (McCammon group) APBS (Baker, McCammon, Holst) Atomistic brownian dynamics simulations with program SDA (Gabdoulline & Wade, 1997) Simulation parameters: time step 2 ps – 10 ps translat. diffusion constant0.02 Å 2 ps -1 rotational diffusion constant radian 2 ps -1 Radius b115 Å Radius c540 Å for each system runs Flöck & Helms, Biophys.J. 87, 65 (2004)

22 9. Vorlesung WS 2004/05Softwarewerkzeuge22 Iteration Algorithm Generalized force vector Diffusion matrices Random displacements Generalized coordinate vector Dickinson, E., Allison, S.A. and McCammon, J.A. (1985) J. Chem. Soc. Farad. Trans. 2 81, 591

23 9. Vorlesung WS 2004/05Softwarewerkzeuge23 fulfil 1, 2, or 3 contact pairs among D156:K79D135:K86 A259:K73D135:K86 S124:K86Y122:G84 naked wild-type COX : horse heart cyt c 140mM exp. rate with solubilized COX Flöck & Helms, Biophys.J. 87, 65 (2004)

24 9. Vorlesung WS 2004/05Softwarewerkzeuge24 Association of horse heart cyt c and naked COX at 140mM. Data for 3 reaction criteria. Nice agreement with exp. trends! Simulated mutation effects on on-rates wild-type COX (3.7) N160D (2.8) D135N (0.3) Flöck & Helms, Biophys.J. 87, 65 (2004)

25 9. Vorlesung WS 2004/05Softwarewerkzeuge25 Simulated ionic strength effects on on-rates Association of P.d. cyt c 552 and naked wild-type COX. Data for 3 reaction criteria. Nice agreement with exp. trends! 10 mM (4.1) 35 mM (1.5) 200 mM (0.1) Flöck & Helms, Biophys.J. 87, 65 (2004)

26 9. Vorlesung WS 2004/05Softwarewerkzeuge26 model of membrane environment Flöck & Helms, Biophys.J. 87, 65 (2004)

27 9. Vorlesung WS 2004/05Softwarewerkzeuge27 Start diffusion proteins only in spherical cap above COX. Inclusion of Membrane Environment This scheme makes no difference for naked COX: comparison of original and spherical-cap starting positions. Flöck & Helms, Biophys.J. 87, 65 (2004)

28 9. Vorlesung WS 2004/05Softwarewerkzeuge28 association rates with and without membrane environment for horse heart cyt c : COX at 140 mM Large effect! Effect of membrane embedding for horse heart cyt c naked COX with membrane potentials from APBS, UHBD Flöck & Helms, Biophys.J. 87, 65 (2004)

29 9. Vorlesung WS 2004/05Softwarewerkzeuge29 association rates with and without membrane environment for P.d. cyt c 552 : COX at 140 mM Association to naked COX slower than for horse heart cyt c. Small effect of membrane! Physiological relevance? Effect of membrane embedding for P.d. cyt c 552 naked COX with membrane Flöck & Helms, Biophys.J. 87, 65 (2004)

30 9. Vorlesung WS 2004/05Softwarewerkzeuge30 Computational studies: gain insight by switching isolated interactions off all charges on membrane charges off (cyt c 552 ) COX charges off (cyt c 552 ) membrane charges off (cyt c) COX charges off (cyt c) Flöck & Helms, Biophys.J. 87, 65 (2004)

31 9. Vorlesung WS 2004/05Softwarewerkzeuge31 Protein – protein association idealized reaction coordinate G free diffusion protein B enters into protein As zone of electrostatic attraction directed diffusion A and B form encounter complex - electrostatically entangled, - no bound complex to form complex, interfaces need to be desolvated + sidechains oriented bound complex

32 9. Vorlesung WS 2004/05Softwarewerkzeuge32 Barnase:Barstar complex extensively studied by Fersht group barnase is an extracellular ribonuclease, barstar its intracellular inhibitor fast binding k on ~ 10 8 M -1 s -1 high affinity k D ~ M binding stabilized by favorable electrostatic binding energy (Wang et al. 2004) association rates extensively studied by Gabdoulline & Wade (1997) Dong et al. (2003)

33 9. Vorlesung WS 2004/05Softwarewerkzeuge33 Aim of this study Aim: clarify nature of encounter complex Means: statistical analysis of brownian dynamics trajectories

34 9. Vorlesung WS 2004/05Softwarewerkzeuge34 Coordinate frame Center of mass coordinates of second protein Rotational coordinates of second protein

35 9. Vorlesung WS 2004/05Softwarewerkzeuge35 Different definitions of distance variable For global view For local view

36 9. Vorlesung WS 2004/05Softwarewerkzeuge36 how does the encounter state look like? representative structures of the encounter state ensemble black balls: reaction atoms of barstar in crystal structure barnase blue: cd 1-2 red: cd min green: cd avg purple: cd center

37 9. Vorlesung WS 2004/05Softwarewerkzeuge37 Zusammenfassung (1)Charakterisierung von Protein-Protein-Wechselwirkung heutzutage am besten möglich per wissensbasierten Ansätzen (Sequenzhomologie). (2)Energetische Charakterisierung noch schwierig - Problem bei Protein-Protein Docking die beste Lösung zu finden (3)Kinetik kann mittels Brownscher Dynamik charakterisiert werden. Versuche, die 6-dimensionale G-Oberfläche durch Mapping der Trajektorien zu erhalten. So kann man den Encounter-Zustand als Minimum der freien Enthalpie entlang einer geeigneten Reaktionskoordinate beschreiben (4)Evolutionäre Relevanz: können verschiedene Teile der Proteinoberfläche für verschiedene Phasen der Proteinassoziation verantwortlich sein? -Geladene Patches: langreichweitige Attraktion -Hydrophobe Patches: bilden Bindungsinterphase Dann ergibt sich ein evolutionärer Druck auf die ganze Oberfläche, nicht nur auf das Bindungsinterface.


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