DETERMINATION OF INTERNUCLEAR DISTANCES AND THE ORIENTATION OF FUNCTIONAL-GROUPS BY SOLID-STATE NMR - ROTATIONAL RESONANCE STUDY OF THE CONFORMATION OF RETINAL IN BACTERIORHODOPSIN

We have used a new solid-state NMR technique-rotational resonance-to determine both internuclear distances and the relative orientations of chemical groups (dihedral angles) in retinal bound to bacteriorhodopsin (bR) and in retinoic acid model compounds. By matching the rotational resonance condition (delta = n omega(r),/2 pi, where delta is the difference in isotropic chemical shifts for two dipolar coupled spins, omega(T)/2 pi is the mechanical rotational frequency of the sample in the MAS experiment, and n is a small integer denoting the order of the resonance), we selectively reintroduce the dipolar coupling and enhance the rate of magnetization exchange. Spectroscopic data and theoretical simulations of the magnetization exchange trajectories for the 8,18-C-13 dipolar coupled pair in retinoic: acid model compounds, crystallized in both the 6-s-cis and 6-s-trans forms, indicate that an accurate determination of the internuclear distance is possible. For the n = 1 resonance we find the distance determination to be reasonably independent of the relative orientation of the groups. In contrast, for the n = 2 resonance, there is a more pronounced dependence on the relative orientation of the groups which permits an estimate of the angle around the 6-s bond for the cis and trans forms to be 42 +/- 5 degrees and 90 +/- 10 degrees, respectively, in good agreement with crystallography. In bR we demonstrate that the 8-C-13-18-C-13 distance is 4.1 Angstrom and the average 8-C-13-16-C-13/8-C-13-17-C-13 distance is 3.3-3.5 Angstrom. These distance determinations depend somewhat on assumed values for the relaxation processes of the zero-quantum state T-2(ZQ), and the resulting errors are larger than in the model compounds, but probably less than 0.4 Angstrom. The data on bR demonstrate unambiguously that the retinal is in a 6-s-trans conformation, confirming the previous interpretation of C-13 chemical shift data and other measurements. Our work clearly suggests that dihedral angle measurements should also be possible with additional orders of rotational resonance. These studies demonstrate a new and robust method for determining internuclear distances and chemical orientations. The prospects appear very encouraging for measuring C-C distances up to 5.0 or 6.0 Angstrom with an accuracy of better than 0.4 Angstrom in very large enzymes, and in same cases for determining the relative orientation of chemical groups.