The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ((2)H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal (2)H-labeled at the C(5), C(9), or C(13) methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of (2)H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121, 7636-7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the beta-ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state (2)H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations.

SummaryRhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed 2 H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with 2 H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state 2 H NMR spectra were acquired for aligned membranes in the lowtemperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations together with the alignment disorder (mosaic spread). The 2 H NMR structure of 11-cis-retinal in the dark state revealed torsional twisting of the polyene chain and the β-ionone ring. The distorted retinylidene ligand undergoes restricted motion, as evinced by order parameters of ≈ 0.9 for the rapidly spinning C-C 2 H 3 groups, with off-axial fluctuations of ≈ 15°. Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond, which explains its rapid photochemistry and indicates the trajectory of the 11-cis to trans isomerization.For the cryotrapped meta I state, the 2 H NMR structure showed a reduction of the polyene strain, whereas the β-ionone ring maintained its torsional twisting. Strain energy and dynamics of retinal are interpreted with regard to substituent control of receptor activation. Steric hindrance between trans retinal and Trp 265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and classical molecular mechanics simulations, and show how 2 H NMR can be applied to ligands bound to GPCRs in relation to their characteristic mechanisms of action.

In seeking to understand G protein-coupled receptor (GPCR)-mediated signaling, X-ray and magnetic resonance approaches have played important roles—yet neither the protein dynamics nor the plasticity of GPCRs are amenable to study. Here we show that solid-state 2H NMR relaxation elucidates picosecond-nanosecond timescale motions of the retinal ligand that impact upon larger-scale functional dynamics of rhodopsin in membranes. A multiscale activation mechanism is put forward, whereby retinal initiates collective helix fluctuations in the Meta I–Meta II equilibrium on the microsecond-millisecond timescale.