Retinal Conformation and Dynamics in Activation of Rhodopsin Illuminated by Solid-state 2H NMR Spectroscopy

Solid-state NMR spectroscopy gives a powerful avenue for investigating G protein-coupled receptors and other integral membrane proteins in a native-like environment. This article reviews the use of solid-state H-2 NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific H-2 NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability H-2 NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three-fold (C-3) axes with an order parameter for the off-axial motion of S-C3 approximate to 0.9. For the dark state, the H-2 NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the beta-ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11 = C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11-cis to trans isomerization. In addition, H-2 NMR has been applied to study the retinylidene dynamics in the dark and light-activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the beta-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the beta-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state H-2 NMR thus provides information about the flow of energy that triggers changes in hydrogen-bonding networks and helix movements in the activation mechanism of the photoreceptor.