1. Photoactivation of the Photoactive Yellow Protein chemistry department Imperial Colege London London SW7 2AZ United Kingdom 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

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

3. Photoactive Yellow Protein 125 residues 125 residues chromophore chromophore

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

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

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

7. molecular dynamics nuclei are classical particles nuclei are classical particles potential energy and forces potential energy and forces - Newton’s equation of motion - molecular mechanics - numerically integrate eom - quantum mechanics

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

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

10. excited state quantum chemistry Hartree-Fock approximation for ground state Hartree-Fock approximation for ground state all are optimized (self-consistent field) all are optimized (self-consistent field) no static/dynamic correlation no static/dynamic correlation 2n electrons

11. excited state quantum chemistry Complete Active Space SCF Complete Active Space SCF and are optimized simultaneously and are optimized simultaneously static correlation static correlation excited states excited states or

12. excited state quantum chemistry simple (but incorrect) CAS expansion simple (but incorrect) CAS expansion ground state: ground state: excited state: excited state: optimize 2 nd root optimize 2 nd root

13. electronic transitions in QM/MM diabatic surface hopping diabatic surface hopping - t 1 : - t 2 : - t 3 : - t 4 : - swap electronic states

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

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

16. results photo-isomerization photo-isomerization

17. results

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

19. results unsuccessful photo-isomerization unsuccessful photo-isomerization

20. results comparison with experiment comparison with experiment - fluorescence lifetime - quantum yield ~0.3 (exp. 0.35) ~0.3 ps (exp. 0.43/4.8 ps) - S 1 -S 0 gap oscillations 1.6 and 4.8 10 12 Hz (exp. 1.5 and 4.2 10 12 Hz)

21. results preferential stabilization of S 1 in PYP preferential stabilization of S 1 in PYP

22. - twisted S 1 minimum geometry in PYP results preferential stabilization of S 1 in PYP preferential stabilization of S 1 in PYP - charge distribution in S 0 and S 1

23. results preferential stabilization of S 1 in PYP preferential stabilization of S 1 in PYP - electrostatic interaction with Arginine 52 - conical intersection geometry in PYP

24. results meta-stable pR intermediate (continued) meta-stable pR intermediate (continued)

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

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

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

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

29. conclusions signal transduction signal transduction - signal transduction in the cell - proton transfer from Glu 46 - partial unfolding - destabilization

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