Three electronic state model of the primary phototransformation of bacteriorhodopsin.

The primary all-trans --> 13-cis photoisomerization of retinal in bacteriorhodopsin has been investigated by means of quantum chemical and combined classical/quantum mechanical simulations employing the density matrix evolution method. Ab initio calculations on an analog of a protonated Schiff base of retinal in vacuo reveal two excited states S1 and S2, the potential surfaces of which intersect along the reaction coordinate through an avoided crossing, and then exhibit a second, weakly avoided, crossing or a conical intersection with the ground state surface. The dynamics governed by the three potential surfaces, scaled to match the in situ level spacings and represented through analytical functions, are described by a combined classical/quantum mechanical simulation. For a choice of nonadiabatic coupling constants close to the quantum chemistry calculation results, the simulations reproduce the observed photoisomerization quantum yield and predict the time needed to pass the avoided crossing region between S1 and S2 states at tau1 = 330 fs and the S1 --> ground state crossing at tau2 = 460 fs after light absorption. The first crossing follows after a 30 degrees torsion on a flat S1 surface, and the second crossing follows after a rapid torsion by a further 60 degrees. tau1 matches the observed fluorescence lifetime of S1. Adjusting the three energy levels to the spectral shift of D85N and D212N mutants of bacteriorhodospin changes the crossing region of S1 and S2 and leads to an increase in tau1 by factors 17 and 10, respectively, in qualitative agreement with the observed increase in fluorescent lifetimes.

Molecular dynamics study of the 13-cis form (bR548) of bacteriorhodopsin and its photocycle.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.

Molecular dynamics study of bacteriorhodopsin and artificial pigments.

The structure of bacteriorhodopsin based on electron microscopy (EM) studies, as provided in Henderson et al. (1990), is refined using molecular dynamics simulations. The work is based on a previously refined and simulated structure which had added the interhelical loops to the EM model of bR. The present study applies an all-atom description to this structure and constraints to the original Henderson model, albeit with helix D shifted. Sixteen waters are then added to the protein, six in the retinal Schiff base region, four in the retinal-Asp-96 interstitial space, and six near the extracellular side. The root mean square deviation between the resulting structure and the Henderson et al. (1990) model measures only 1.8 A. Further simulations of retinal analogues for substitutions at the 2- and 4-positions of retinal and an analogue without a beta-ionone ring agree well with observed spectra. The resulting structure is characterized in view of bacteriorhodopsin's function; key features are (1) a retinal Schiff base-counterion complex which is formed by a hydrogen bridge network involving six water molecules, Asp-85, Asp-212, Tyr-185, Tyr-57, Arg-82, and Thr-89, and which exhibits Schiff base nitrogen-Asp-85 and -Asp-212 distances of 6 and 4.6 A; (2) retinal assumes a corkscrew twist as one views retinal along its backbone; and (3) a deviation from the usual alpha-helical structure of the cytoplasmic side of helix G.