Structural Investigation of the Active Site in Bacteriorhodopsin: Geometric Constraints on the Roles of Asp-85 and Asp-212 in the Proton-Pumping Mechanism from Solid State NMR

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Abstract
Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR555, bR568, and M412 states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via 13C−13C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 ± 0.6 and 4.8 ± 1.0 Å for the [4-13C]Asp-212 to [14-13C]retinal distances in bR568 and M412, respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 ± 10% upon conversion to the M-state. The absence of cross-peaks from [14-13C]retinal to [4-13C]Asp-85 in all states and between any [4-13C]Asp residue and [14-13C]retinal in bR555 indicates that these distances exceed 6.0 Å. For bR568, the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.