¹ H and ¹³ C MAS NMR evidence for pronounced ligand–protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin

Abstract

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven ␣-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete 1 H and 13 C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly 13 C-labeled 11-cis-retinal. From the labeled pigment, 13 C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment.

Computational Studies of the Primary Phototransduction Event in Visual Rhodopsin

“…Such a 6s-cis structure is characterized by a substantial negative twist of the dihedral angle (-44°) at the C6-C7 bond. 1 These theoretical results are partially consistent with recent NMR studies 43,44 and with the analysis of the recently resolved X-ray structure at 2.2 Å resolution. 5 7.3.…”

“…An important aspect addressed by recent DFT QM/MM simulations 2 is the observed upfield shift of σ C at C5, which has been previously assigned to the interaction between C5 and the carboxylic group of Glu-122. 43 However, QM/ MM calculations indicate that the electrostatic influence of Glu-122 is negligible when such a residue is protonated as suggested by FTIR experiments. 49 In contrast, QM/MM studies found that the most significant upfield shift on C5 is due to Trp-265, with an upfield effect of -2.0 ppm.…”

“…The overall agreement between theoretical and experimental data is quite satisfactory, specially considering that 13 C NMR chemical shifts are often reported with errors as large as (1 ppm and larger when different studies are compared. 33,43 The theoretical predictions, however, seem to underestimate changes in chemical shifts of C17 and C20, due to an overestimation of the chemical shifts of the corresponding methyl-substituent groups in rhodopsin. An important aspect addressed by recent DFT QM/MM simulations 2 is the observed upfield shift of σ C at C5, which has been previously assigned to the interaction between C5 and the carboxylic group of Glu-122.…”

“…In particular, NMR studies by Watts and co-workers, 41 as well as theoretical work by Birge and coworkers, 42 concluded that the chromophore geometry is 6s-trans at the C6-C7 bond, in contrast to the 6s-cis conformation assumed by earlier NMR and optical studies. 33,43,[45][46][47][48][49][50] To address the orientation of the -ionone ring, according to rigorous DFT QM/MM calculations, a computational model of the 6s-trans isomer has been constructed by geometry relaxation of the system after rotation of the -ionone ring around the C6-C7 bond. 2 The resulting 6s-trans structure, obtained at the ONIOM electronicembedding (B3LYP/6-31G*:Amber) level of theory, was found to be as stable as the 6s-cis isomer for many thermally accessible configurations and about 8 kcal/mol less stable than the 6s-cis structure when minimum energy geometries are compared.…”

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

“…However, the lightactivated Meta II state is transient and crystal deformation occurs giving low-resolution data (14). Crystal structures of ligand-free opsin (19) with a bound synthetic G t peptide (20) show an elongation of the protein (21, 22) due to helical displacements (19)-but opsin does not contain the activating all-trans-retinal ligand, and its activity does not match rhodopsin (20).Spectroscopic approaches including spin-label EPR (23), 13 C NMR (18,(24)(25)(26), and Fourier transform infrared (FTIR) (17, 27) studies are thus needed to establish the activation mechanism of the photoreceptor as it underlies the visual process. Solid-state NMR spectroscopy (28) is particularly important, as it gives knowledge of both protein structure and dynamics in a membrane lipid environment (29).…”