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Photoactivation of the Photoactive Yellow Protein

Photoactivation of the Photoactive Yellow Protein. Gerrit Groenhof , Berk Hess, Marc F. Lensink, Mathieu Bouxin-Cademartory, Sam de Visser Massimo Olivucci, Herman J.C. Berendsen, Alan E. Mark and Michael A. Robb. dept. of biophysical chemistry University of Groningen

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Photoactivation of the Photoactive Yellow Protein

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  1. Photoactivation of the Photoactive Yellow Protein Gerrit Groenhof, Berk Hess, Marc F. Lensink, Mathieu Bouxin-Cademartory, Sam de Visser Massimo Olivucci, Herman J.C. Berendsen, Alan E. Mark and Michael A. Robb dept. of biophysical chemistry University of Groningen Nijenborg 4, 9747 AG Groningen The Netherlands chemistry department Imperial Colege London London SW7 2AZ United Kingdom

  2. Photoactive Yellow Protein • cytoplasmic photoreceptor • Halorhodospira halophila • negative photo-tactic response to blue light

  3. Photoactive Yellow Protein • 125 residues • chromophore

  4. Photoactive Yellow Protein • photocycle - photon absorption - isomerization (ns) - part. unfolding (ms) - relaxation (ms)

  5. aims • to understand how • photon absorption induces isomerization • of the chromophore inside the protein • isomerization of the chromophore induces • structural changes in the protein and leads • to signalling • the protein mediates these processes

  6. photo-chemistry • ground-state vs. excited-state reactivity - transition state - surface crossing - dynamics govern rate - statistics govern rate

  7. molecular dynamics • nuclei are classical particles - Newton’s equation of motion - numerically integrate e.o.m. • potential energy and forces - molecular mechanics forcefield (MM) - molecular quantum mechanics (QM)

  8. quantum mechanics • solving electronic Schrödinger equation • potential field for nuclei • more accurate than forcefield - excited states, transitions between el. states - bond breaking/formation • computationally demanding

  9. QM/MM hybrid model • QM subsystem embedded in MM system A. Warshel & M. Levitt. J. Mol. Biol.103: 227-249 (1976)

  10. simulation setup • QM/MD simulation of PYP - dodecahedron with one protein molecule - 5089 water molecules (SPC) - 6 Na+ ions

  11. simulation setup • QM subsystem - chromophore (22 atoms) - CASSCF accurate ground and excited states of (small) molecules - diabatic surface hopping transitions between ground and excited states • MM subsystem - apo protein, water & ions (16526 atoms) - gromos96 force-field

  12. results • photo-isomerization

  13. results • comparison with experiment - crystal structure of the intermediate state (pR) R. Kort et al. J. Biol. Chem.279: 26417-26424 (2004)

  14. results • unsuccessful photo-isomerization

  15. results • comparison with experiment - quantum yield ~0.3 (exp. 0.35) - S1-S0 gap oscillations 1.6 and 4.8 1012Hz (exp. 1.5 and 4.2 1012 Hz) - fluorescence lifetime ~0.3 ps (exp. 0.43/4.8 ps)

  16. results • preferential stabilization of S1 in PYP

  17. results • preferential stabilization of S1 in PYP - twisted S1 minimum geometry in PYP - charge distribution in S0 and S1

  18. results • preferential stabilization of S1 in PYP - conical intersection geometry in PYP - electrostatic interaction with Arginine 52

  19. results • meta-stable pR intermediate (continued)

  20. results • after photo-isomerization - classical MD simulation (Gromos96) - protein remains stable - no signalling, isomerization alone is not sufficient

  21. results • proton transfer - QM/MM analysis (PM3/Gromos96) QM system - before isomerization proton transfer not possible - after isomerization proton transfer possible from glutamic acid

  22. results • after proton transfer - classical MD simulation (Gromos96) - conformational changes - increased flexibility in N-terminus - agreement with NMR data

  23. conclusions • isomerization mechanism - on S1, double bond rotates to 90° - rotation does not cause transition to S0 • rather, bond stretching causes transtion to S0 • on S0, strain disrupts H-bond with bb amide

  24. conclusions • signal transduction - proton transfer from Glu46 - destabilization • partial unfolding • signal transduction in the cell

  25. acknowledgements Jocelyne Vreede & Klaas Hellingwerf University of Amsterdam Amsterdam, The Netherlands Haik Chosrowjan & Noboru Mataga University of Osaka Osaka, Japan Michael Klene & Valerio Trigari King’s College/Imperial College London, United Kingdom

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