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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 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
Photoactive Yellow Protein • cytoplasmic photoreceptor • Halorhodospira halophila • negative photo-tactic response to blue light
Photoactive Yellow Protein • 125 residues • chromophore
Photoactive Yellow Protein • photocycle - photon absorption - isomerization (ns) - part. unfolding (ms) - relaxation (ms)
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
photo-chemistry • ground-state vs. excited-state reactivity - transition state - surface crossing - dynamics govern rate - statistics govern rate
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)
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
QM/MM hybrid model • QM subsystem embedded in MM system A. Warshel & M. Levitt. J. Mol. Biol.103: 227-249 (1976)
simulation setup • QM/MD simulation of PYP - dodecahedron with one protein molecule - 5089 water molecules (SPC) - 6 Na+ ions
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
results • photo-isomerization
results • comparison with experiment - crystal structure of the intermediate state (pR) R. Kort et al. J. Biol. Chem.279: 26417-26424 (2004)
results • unsuccessful photo-isomerization
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)
results • preferential stabilization of S1 in PYP
results • preferential stabilization of S1 in PYP - twisted S1 minimum geometry in PYP - charge distribution in S0 and S1
results • preferential stabilization of S1 in PYP - conical intersection geometry in PYP - electrostatic interaction with Arginine 52
results • meta-stable pR intermediate (continued)
results • after photo-isomerization - classical MD simulation (Gromos96) - protein remains stable - no signalling, isomerization alone is not sufficient
results • proton transfer - QM/MM analysis (PM3/Gromos96) QM system - before isomerization proton transfer not possible - after isomerization proton transfer possible from glutamic acid
results • after proton transfer - classical MD simulation (Gromos96) - conformational changes - increased flexibility in N-terminus - agreement with NMR data
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
conclusions • signal transduction - proton transfer from Glu46 - destabilization • partial unfolding • signal transduction in the cell
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