The resonance Raman spectrum of the 11-cis retinal protonated Schiff base chromophore in rhodopsin exhibits low-frequency normal modes at 93, 131, 246, 260, 320, 446, and 568 cm -1 . Their relatively strong Raman activities reveal that the photoexcited chromophore undergoes rapid nuclear motion along torsional coordinates that may be involved in the 200-fs isomerization about the C 11 dC 12 bond. Resonance Raman spectra of rhodopsins regenerated with isotopically labeled retinal derivatives and demethyl retinal analogues were obtained in order to determine the vibrational character of these low-frequency modes and to assign the C 11 dC 12 torsional mode. 13 C substitutions of atoms in the C 12 -C 13 or C 13 dC 14 bond cause the 568-cm -1 mode to shift by ∼8 cm -1 , and deuteration of the C 11 dC 12 bond downshifts the 568-and 260-cm -1 modes by ∼35 and 5 cm -1 , respectively. The magnitudes of these shifts are consistent with those calculated for modes containing significant C 11 dC 12 torsional character. Thus, we assign the 568-cm -1 mode to a localized C 11 dC 12 torsion and the 260-cm -1 mode to a more delocalized torsional vibration involving coordinates from C 10 to C 13 . Consistent with these assignments, these two modes are not Raman active in 13-demethyl, 11-cis rhodopsin which has a planar C 10 ‚‚‚C 13 geometry. Furthermore, the relative Raman scattering strengths of the 260-and 568-cm -1 modes are ∼2-fold higher with preresonant excitation. These data quantitate the instantaneous torsional dynamics of the chromophore about its C 11 dC 12 bond on the S 1 surface and indicate that the isomerization process is facilitated by vibronic coupling of the S 1 and S 2 surfaces via C 11 dC 12 torsional distortion, which reduces the excited-state barrier along the reaction trajectory. We have also examined the low-frequency Raman spectrum of the trans primary photoproduct, bathorhodopsin, and discuss the relevance of its low-frequency torsional modes at ∼54, 92, 128, 151, 262, 276, 324, and 376 cm -1 to the observed femtosecond photochemical dynamics.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.

We describe magic-angle-spinning NMR methods for the accurate determination of internuclear dipole-dipole couplings between homonuclear spins-(1/2) in the solid state. The new sequences use symmetry principles to treat the effect of magic-angle sample-rotation and resonant radio frequency fields. The pulse-sequence symmetries generate selection rules which reduce the interference of undesirable interactions and improve the robustness of the pulse sequences with respect to chemical shift anisotropies. We show that the pulse sequences may be used to estimate distances between 13C spins in organic solids, including bond lengths in systems with large chemical shift anisotropies, such as conjugated systems. For bond-length measurements, the precision of the method is +/-2 pm with a systematic overestimate of the internuclear distance by 3 +/- 1 pm. The method is expected to be a useful tool for investigating structural changes in macromolecules.