Computational Studies of the Photoreceptor Rhodopsin Scott E. Feller Wabash College
Rhodopsin Photocycle Dark-adapted Rhodopsin hn Isomerize retinal Photorhodopsin ~200 fs Bathorhodopsin Meta-II ms timescale Meta-I s timescale ns timescale Lumirhodopsin
Challenging Problem Requires Multiple Complimentary Techniques Changes in conformation and protonation of protein Changes in conformation and protonation of retinal ligand Changes in conformation of solvating phospholipids?
Dark state retinal orientation: Combining 2 H NMR and MD simulation NMR on reconstituted oriented bilayers with labeled retinal methyl groups MD simulations on BlueGene
MD simulation reveals fluctuations in retinal torsions Carried out 26 100 ns simulations of rhodopsin in an explicit lipid bilayer using different random lipid conformations and positions as starting points As expected the conjugated polyene chain is stiff with all dihedrals in cis or trans conformations in all simulations
Dynamic Structure of Retinylidene Ligand of Rhodopsin Probed by Molecular Simulations, J. Mol. Biol. 372, 906-917 MD simulation reveals fluctuations in b-ionone ring orientation Isomerization of C5-C6-C7-C8 dihedral is slow on MD timescale but multiple conformers observed Pooled distribution of conformers from 2.6 s gives excellent agreement with NMR
Modeling the Meta-I state MI formation involves isomerization of retinal and may involve changes in protein protonation state in the binding pocket Carried out simulations (> 1 s each) of competing mechanisms suggested by vibrational spectroscopy experiments Counterion switch Yan, E. et al PNAS 2003, 100, 9262 Complex counterion - Lüdeke, S. et al. J. Mol. Biol. 2005, 353, 345
Counterion switch Compare MD with 2 H NMR Complex counterion Retinal Counterion Switch Mechanism in Vision Evaluated by Molecular Simulations, J. Am. Chem. Soc 28; 16502-16503.
b-ionone ring conformation changes upon isomerization of C11=C12 After isomerization of the 11-cis position the C6-C7 dihedral rotates from s-cis to s-trans state Fluctuations to (-) twisted s-cis state occur on the 100 ns scale
Summary of Dark State/MI MD simulations of dark state (11-cis, PSB) reproduce NMR observables After forcing isomerization of C11=C12, the MI state (11- trans, PSB) is produced on the s time scale. MD reproduces NMR provided the correct side chain protonation state is chosen. The b-ionone ring has significant mobility, sampling twisted s-cis conformations (+/- 60) in the dark state and rotating to s-trans in MI
Challenges Modelling the MII state MII formation occurs on the millisecond time scale, inaccessible to atomistic MD simulation Fortunately crystal structures of the MII state have become available CHARMM style force fields were published only for the PSB, inappropriate for the deprotonated retinal of MII We set out to refine force fields for protonated and deprotonated retinal consistent with each other and with the CHARMM force field for lipids and proteins
Retinal force field parameters depend on protonation state Bond lengths are intermediate between single and double bonds in protonated retinal
Retinal force field parameters depend on protonation state Torsional bariers are intermediate between single and double bonds in protonated retinal
Examples of torsional energy surfaces Large differences are observed near the Schiff base Small differences are observed near the b-ionone ring
An surprising result The C6-C7 bond, that defines the orientation of the ring with respect to the polyene chain, goes against the trend Deprotonated surface shows two minima corresponding to +/- twisted s-cis conformers Protonated surface has minima shifted and the appearance of a local minima at s-trans relative energy (kcal/mol) 5 4 3 2 1 Deprotonated Protonated Lower barriers to rotation in protonated (dark, MI) states 0-150 -100-50 0 50 100 150 C5=C6-C7=C8
Proton affinity depends on b-ionone ring orientation Proton affinity (PA) is the difference in absolute gas-phase energies between protonated and neutral species p r o to n a ffin ity (k c a l/m o l) 2 5 2.5 2 5 2 2 5 1.5 2 5 1 PA = E A- - E HA PA is highest for planar 2 5 0.5 conformations, i.e. planar angles favor the 2 5 0 protonated form raising pk A while deviations 2 4 9.5 from planarity make retinal more acidic 2 4 9-1 5 0-1 0 0-5 0 0 50 1 0 0 1 5 0 C 5 = C 6 -C 7 = C 8
pk A of Schiff based depends on b-ionone ring orientation pk A can be computed in a thermodynamic cycle that combines gas phase PA with QM calculation of DG solv in implicit solvent Accurate pka calculations for carboxylic acids using complete basis set and Gaussian-n models combined with CPCM continuum solvation methods MD Liptak & GC Shields, JACS Absolute values of pk A likely shift due to environment but conformation dependent shifts of ~2 units is predicted among thermally accessible states Is this relevant for MI MII transition? pk A 9.5 9 8.5 8 7.5 7 6.5-150 -100-50 0 50 100 150 C5=C6-C7=C8
Simulation suggests fluctuations in MI state correspond to large pk A changes Thermally accessible transitions between s-cis and s-trans involve pk A change of ~ 2 units, i.e. a 100 fold change in equilibrium constant
Potential implications for Rhodopsin activation Potential for reciprocal control where ligand conformational change (11-cis to all-trans) leads to protein conformational change which subsequently shifts the ligand conformation to one that favors deprotonation Implies that ligand-protein interactions change as a function of the Rhodopsin photocycle
Preliminary results on ligand-protein interactions Begin by examining dark state and MII where crystal structures are available Carry out Umbrella Sampling with Hamiltonian Replica exchange using the C5=C6-C7=C8 dihedral as the reaction coordinate First compute potential of mean force in vacuum, repeat in full membrane protein environment Compute DDG between vacuum and protein. Does it change with photocycle?
Preliminary results on ligand-protein interactions The effect of the protein is to change the free energy profile by ~1 kcal/mol in the deprotonated (MII) case
Preliminary results on ligand-protein interactions The s-trans state is destabilized in MII (but not in dark state) consistent with the protein selecting the ligand conformation that favors deprotonation
Preliminary results on ligand-protein interactions We have not computed the PMF for MI but the unbiased simulation suggests that s-trans is stabilized in MI
Summary MD simulations, after validation by experimental observables, can provide an atomic level interpretation of the structures and fluctuations in membrane proteins The retinal ligand is a complicated species requiring careful parameterization to obtain accurate MD simulations of rhodopsin Fluctuations in b-ionone ring conformation may provide a mechanism to tune the Schiff base pk A
Acknowledgements Mike Lu, Shenghuang Zhu, Lu Hong (Wabash College) Michael Brown (University of Arizona) Blake Mertz (West Virginia University) Alan Grossfield (University of Rochester) NSF MCB 0950258