title: Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin.
abstract: 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 (2)H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific (2)H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state (2)H 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 (2)H 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.
authors.affiliation: Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Struts
authors.forename: Gilmar F J
authors.initials: GF
authors.lastname: Salgado
authors.forename: Michael F
authors.initials: MF
authors.lastname: Brown
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D011487:Protein Conformation; D012172:Retinaldehyde / Q000528:radiation effects*; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins; D012243:Rhodopsin; D012172:Retinaldehyde