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 d etermine both internuclear distances and the relative orientations of chemical groups (dihedral angles) in retinal bound to bacteriorhodopsi n (bR) and in retinoic acid model compounds. By matching the rotationa l resonance condition (delta = n omega(r),/2 pi, where delta is the di fference in isotropic chemical shifts for two dipolar coupled spins, o mega(T)/2 pi is the mechanical rotational frequency of the sample in t he MAS experiment, and n is a small integer denoting the order of the resonance), we selectively reintroduce the dipolar coupling and enhanc e the rate of magnetization exchange. Spectroscopic data and theoretic al simulations of the magnetization exchange trajectories for the 8,18 -C-13 dipolar coupled pair in retinoic: acid model compounds, crystall ized in both the 6-s-cis and 6-s-trans forms, indicate that an accurat e determination of the internuclear distance is possible. For the n = 1 resonance we find the distance determination to be reasonably indepe ndent 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 arou nd the 6-s bond for the cis and trans forms to be 42 +/- 5 degrees and 90 +/- 10 degrees, respectively, in good agreement with crystallograp hy. In bR we demonstrate that the 8-C-13-18-C-13 distance is 4.1 Angst rom 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 val ues for the relaxation processes of the zero-quantum state T-2(ZQ), an d the resulting errors are larger than in the model compounds, but pro bably less than 0.4 Angstrom. The data on bR demonstrate unambiguously that the retinal is in a 6-s-trans conformation, confirming the previ ous interpretation of C-13 chemical shift data and other measurements. Our work clearly suggests that dihedral angle measurements should als o be possible with additional orders of rotational resonance. These st udies demonstrate a new and robust method for determining internuclear distances and chemical orientations. The prospects appear very encour aging for measuring C-C distances up to 5.0 or 6.0 Angstrom with an ac curacy of better than 0.4 Angstrom in very large enzymes, and in same cases for determining the relative orientation of chemical groups.