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 inter helical 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 Å. Further simulations of retinal analogues for substitutions at the 2- and 4-positions of retinal and an analogue without a β-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 α-helical structure of the cytoplasmic side of helix G.
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