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

Abstract

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 2 H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific 2 H 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 2 H 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 % 0:9: For the dark state, the 2 H NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the b-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, 2 H 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 b-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the b-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state 2 H 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.

Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

“…3b as a torsional fluctuation while the peak at 65 cm −1 is associated with a chain torsion coupled with a retinal ring bending motion (Supplementary Figure S6 and Table 1). Both modes (at 65 and 85 cm −1 ) are related to rhodopsin in the dominant (inactive-type) conformation where both the polyene chain and the β-ionone ring are known to undergo constrained torsional twisting46. Likewise, the peak close to 40 cm −1 in the simulation spectrum of the 11- cis retinal in Fig.…”

Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy

“…Solid-state NMR has been extensively applied to investigations of rhodopsin including magic-angle spinning 13 C NMR [25, 26, 65, 128–130], 15 N NMR [131] and 2 H NMR [24, 52, 53, 56, 132]. Both the protein as well as the bound retinal ligand have been investigated with solid-state 13 C NMR of rhodopsin in detergent micelles [25, 26, 130].…”

“…Both the protein as well as the bound retinal ligand have been investigated with solid-state 13 C NMR of rhodopsin in detergent micelles [25, 26, 130]. Complementary 2 H NMR studies of the retinal cofactor of rhodopsin in a native-like membrane invironment have also been conducted [24, 52, 53, 56, 132]. The 2 H NMR structure of 11- cis retinal in the dark state reveals dihedral twisting of the polyene chain and the β-ionone ring, in contrast to microbial rhodopsins such as bacteriorhodopsin.…”

“…Second, membrane proteins are studied in a native-like membrane environment [20, 21, 25, 49–54] where protein function is preserved [37, 55]. Last, solid-state NMR relaxation experiments reveal dynamical knowledge for non-crystalline biomembrane specimens that cannot be obtained with X-ray crystallography or other methods [56]. It is mainly theoretical molecular dynamics (MD) simulations that provide such comprehensive dynamical information at present [33], which require validation through experimental studies as described here.…”

Structure and function of G protein-coupled receptors using NMR spectroscopy

“…Deuterium NMR methods have been used extensively for studying the orientation and dynamics of the retinal chromophore in dark rhodopsin [170,171] and its photointermediates [172–174]. The strategy in these experiments is outlined in Fig.…”