Photoactivation of the Photoactivation of the Photoactive Yellow ProteinPhotoactive Yellow Protein

chemistry departmentchemistry departmentImperial Colege LondonImperial Colege LondonLondon SW7 2AZLondon SW7 2AZUnited KingdomUnited Kingdom

Gerrit GroenhofGerrit Groenhof, Berk Hess, Marc F. Lensink,, Berk Hess, Marc F. Lensink,Mathieu Bouxin-Cademartory, Sam de VisserMathieu Bouxin-Cademartory, Sam de VisserMassimo Olivucci, Massimo Olivucci, Herman J.C. Berendsen, Herman J.C. Berendsen, Alan E. Mark and Alan E. Mark and Michael A. RobbMichael A. Robb

dept. of biophysical chemistrydept. of biophysical chemistryUniversity of GroningenUniversity of GroningenNijenborg 4, 9747 AG GroningenNijenborg 4, 9747 AG GroningenThe NetherlandsThe Netherlands

Photoactive Yellow ProteinPhotoactive Yellow Protein

• cytoplasmic photoreceptorcytoplasmic photoreceptor

• HalorhodospiraHalorhodospira halophila halophila

• negative photo-tactic response to blue lightnegative photo-tactic response to blue light

Photoactive Yellow ProteinPhotoactive Yellow Protein

• 125 residues125 residues

• chromophorechromophore

Photoactive Yellow ProteinPhotoactive Yellow Protein

• photocyclephotocycle

-- isomerization (ns)isomerization (ns)

-- part. unfolding (part. unfolding (s)s)

-- relaxation (ms)relaxation (ms)

-- photon absorptionphoton absorption

- photon absorption induces isomerizationphoton absorption induces isomerization of the chromophore inside the proteinof the chromophore inside the protein

aimsaims

• to understand howto understand how

- isomerization of the chromophore inducesisomerization of the chromophore induces structural changes in the protein and leadsstructural changes in the protein and leads to signallingto signalling

- the protein mediates these processesthe protein mediates these processes

photo-chemistryphoto-chemistry

• ground-state ground-state vsvs. excited-state reactivity. excited-state reactivity

- transition state - transition state - surface crossing- surface crossing

- statistics govern rate - statistics govern rate - dynamics govern rate- dynamics govern rate

molecular dynamicsmolecular dynamics• nuclei are classical particlesnuclei are classical particles

NRnnn xxxVxmFn

,,, 21

• potential energy and forcespotential energy and forces

k

kkN pxvxxxV ;,,, 21

202

21

000 ttxtttxtxtx nnnn

- Newton’s equation of motion- Newton’s equation of motion

- molecular mechanics forcefield (MM)- molecular mechanics forcefield (MM)

dxxxHxxxV eNeN ,,,ˆ,,, 2121

- numerically integrate e.o.m.- numerically integrate e.o.m.

- molecular quantum mechanics (QM)- molecular quantum mechanics (QM)

quantum mechanicsquantum mechanics

• solving electronic Schrsolving electronic Schröödinger equationdinger equation

ee EH ˆ

• potential field for nuclei potential field for nuclei

dHF eeRn n

ˆ

• more accurate than forcefieldmore accurate than forcefield

• computationally demanding computationally demanding

- excited states, transitions between el. states- excited states, transitions between el. states

- bond breaking/formation- bond breaking/formation

QM/MM hybrid modelQM/MM hybrid model

• QMQM subsystem embedded in subsystem embedded in MMMM system systemA. Warshel & M. Levitt. A. Warshel & M. Levitt. J. Mol. Biol.J. Mol. Biol. 103103: 227-249 (1976): 227-249 (1976)

simulation setupsimulation setup• QM/MD simulation of PYPQM/MD simulation of PYP

- dodecahedron- dodecahedron

- 5089 water molecules (SPC)- 5089 water molecules (SPC)

- 6 Na+ ions- 6 Na+ ions

with one protein moleculewith one protein molecule

simulation setupsimulation setup• QM subsystemQM subsystem

- - diabatic diabatic surface hoppingsurface hopping

- chromophore (22 atoms)- chromophore (22 atoms)

- CASSCF- CASSCF

transitions between ground and excited statestransitions between ground and excited states

- apo protein, water & ions (16526 atoms)- apo protein, water & ions (16526 atoms)

- gromos96 force-field- gromos96 force-field

• MM subsystemMM subsystem

accurate ground and excited states of (small) moleculesaccurate ground and excited states of (small) molecules

resultsresults• photo-isomerizationphoto-isomerization

resultsresults• comparison with experimentcomparison with experiment

- crystal structure of the intermediate state (pR)- crystal structure of the intermediate state (pR)

R. Kort R. Kort et alet al. . J. Biol. Chem.J. Biol. Chem. 279279: 26417-26424 (2004): 26417-26424 (2004)

resultsresults• unsuccessful photo-isomerizationunsuccessful photo-isomerization

resultsresults• comparison with experimentcomparison with experiment

- fluorescence lifetime- fluorescence lifetime

- quantum yield- quantum yield~0.3 (exp. 0.35)~0.3 (exp. 0.35)

~0.3 ps (exp. 0.43/4.8 ~0.3 ps (exp. 0.43/4.8 ps)ps)

- S- S11-S-S00 gap oscillations gap oscillations1.6 and 4.8 101.6 and 4.8 101212Hz Hz (exp. 1.5 and 4.2 10(exp. 1.5 and 4.2 101212 Hz) Hz)

resultsresults

• preferential stabilization of Spreferential stabilization of S11 in PYP in PYP

- twisted S- twisted S11 minimum geometry in PYP minimum geometry in PYP

resultsresults

• preferential stabilization of Spreferential stabilization of S11 in PYP in PYP

- charge distribution in S- charge distribution in S00 and S and S11

resultsresults

• preferential stabilization of Spreferential stabilization of S11 in PYP in PYP

- electrostatic interaction with Arginine 52- electrostatic interaction with Arginine 52

- conical intersection geometry in PYP- conical intersection geometry in PYP

resultsresults• meta-stable pR intermediate (continued)meta-stable pR intermediate (continued)

resultsresults• after photo-isomerizationafter photo-isomerization

- protein remains stable- protein remains stable

- no signalling, isomerization alone is not sufficient- no signalling, isomerization alone is not sufficient

- classical MD simulation (Gromos96)- classical MD simulation (Gromos96)

resultsresults• proton transferproton transfer

- before isomerization- before isomerization

- after isomerization- after isomerization

proton transfer not possibleproton transfer not possible

proton transfer possible from glutamic acidproton transfer possible from glutamic acid

- QM/MM analysis (PM3/Gromos96)- QM/MM analysis (PM3/Gromos96)

QM system

resultsresults• after proton transferafter proton transfer

- conformational changes- conformational changes

- increased flexibility in N-terminus- increased flexibility in N-terminus

- agreement with NMR data- agreement with NMR data

- classical MD simulation (Gromos96)- classical MD simulation (Gromos96)

conclusionsconclusions• isomerization mechanismisomerization mechanism

- on S0, strain disrupts H-bond with bb amideon S0, strain disrupts H-bond with bb amide

- on S1, double bond rotates to 90- on S1, double bond rotates to 90°°

- rather, bond stretching causes transtion to S0rather, bond stretching causes transtion to S0

- rotation does not cause transition to S0- rotation does not cause transition to S0

conclusionsconclusions• signal transductionsignal transduction

- signal transduction in the cellsignal transduction in the cell

- proton transfer from Glu- proton transfer from Glu4646

- partial unfoldingpartial unfolding

- destabilization- destabilization

acknowledgementsacknowledgements

Jocelyne Vreede & Klaas HellingwerfJocelyne Vreede & Klaas Hellingwerf

Haik Chosrowjan & Noboru MatagaHaik Chosrowjan & Noboru Mataga

Michael Klene & Valerio Trigari Michael Klene & Valerio Trigari

University of AmsterdamUniversity of AmsterdamAmsterdam, The NetherlandsAmsterdam, The Netherlands

University of OsakaUniversity of OsakaOsaka, JapanOsaka, Japan

King’s College/Imperial CollegeKing’s College/Imperial CollegeLondon, United KingdomLondon, United Kingdom