Solid-state ²H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin

Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state ²H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific ²H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state ²H NMR relaxation (spin-lattice, T_1Z, and quadrupolar-order, T_1Q) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation - even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the ²H NMR data suggest the β-ionone ring remains in its hydrophobic binding pocket in all three states of the protein. We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the β-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response - rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.