Insight into the chromophore of rhodopsin and its Meta-II photointermediate by 19F solid-state NMR and chemical shift tensor calculations.

19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local environment. In this study we have exploited a fluorine substituent, strategically placed at the C-12-position of 11-cis retinal, the chromophore of visual rhodopsins. This label was used to explore the local environment of the chromophore in the ground state of bovine rhodopsin and its active photo-intermediate Meta II. In addition, the chemical shift and tensor elements of the chromophore in the free state in a membrane environment and the bound state in the protein were determined. Upon binding of the chromophore into rhodopsin and Meta II, the isotropic chemical shift changes in the opposite direction by +9.7 and -8.4 ppm, respectively. An unusually large isotropic shift difference of 35.9 ppm was observed between rhodopsin and Meta II. This partly originates in the light-triggered 11-cis to all-trans isomerization of the chromophore. The other part reflects the local conformational rearrangements in the chromophore and the binding pocket. These NMR data were correlated with the available X-ray structures of rhodopsin and Meta II using bond polarization theory. For this purpose hydrogen atoms have to be inserted and hereto a family of structures were derived that best correlated with the well-established 13C chemical shifts. Based upon these structures, a 12-F derivative was obtained that best corresponded with the experimentally determined 19F chemical shifts and tensor elements. The combined data indicate strong changes in the local environment of the C-12 position and a substantially different interaction pattern with the protein in Meta II as compared to rhodopsin.

Fluoro derivatives of retinal illuminate the decisive role of the C(12)-H element in photoisomerization and rhodopsin activation.

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it can be transformed into the all-trans isomer that acts as a full agonist. This photoisomerization process is extremely efficient: 2 out of 3 photons are effective, full stereoselectivity is achieved, and stereoinversion occurs within 200 fs. The mechanism behind this process is still not really understood, although the available evidence points at the twisted C(9)-C(13) segment of the 11-cis ligand as the quintessence. To further dissect the role of this segment, we have generated the 10-fluoro, 12-fluoro, and 14-fluoro analogues of rhodopsin. A fluoro substituent brings in only little more volume than hydrogen, but considerably more mass and polarizability. The analogue pigments were compared to rhodopsin with respect to their photosensitivity (quantum yield), light-induced structural transitions (UV-vis and FT-IR spectroscopy), and signaling activity (G protein activation rate). We find that 14-F substitution is quite neutral, while 10-F and 12-F substitutions exert significant but distinct effects. The 10-F pigment exhibits a quantum yield similar to that of rhodopsin (0.65) but strongly perturbed thermodynamics of the structural transitions following photoactivation and only 20% of the native signaling activity. The 12-F pigment exhibits a significantly decreased quantum yield (0.47) and signaling activity (30%) but mixed effects on the structural transitions. These properties are compared to those of the corresponding methyl derivatives. We conclude that rotation of the C(12)-H bond of the rhodopsin chromophore is a major rate-limiting factor in the photoisomerization process, while the C(10)-H moiety plays a dominant role in ligand relaxation and further rearrangements following photoactivation.