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

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 estab lished for the bR(555), bR(568), and M-412 states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via C-13 -C-13 magnetization exchange, which was observed in two-dimensional RF-driv en recoupling (RFDR) and spin diffusion experiments. A comparison of relati ve RFDR cross-peak intensities with simulations of the NMR experiments yiel ds distance measurement and 4.4 +/- 0.6 and 4.8 +/- 1.0 Angstrom for the [1 4-C-13]retinal distances in bR(568) and M-412, respectively(,) The spin dif fusion 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-C-13]retinal to [4-C-13]Asp-85 i n all states and between any [4-C-13]Asp residue and [14-C-13]retinal in bR (555) indicates that these distances exceed 6.0 Angstrom. For bR(568) the N MR distance constraints are in agreement with the results fi om recent diff raction 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.