Structural Analysis and Dynamics of Retinal Chromophore in Dark and Meta I States of Rhodopsin from 2H NMR of Aligned Membranes

Abstract

SummaryRhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed 2 H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with 2 H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state 2 H NMR spectra were acquired for aligned membranes in the lowtemperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations together with the alignment disorder (mosaic spread). The 2 H NMR structure of 11-cis-retinal in the dark state revealed torsional twisting of the polyene chain and the β-ionone ring. The distorted retinylidene ligand undergoes restricted motion, as evinced by order parameters of ≈ 0.9 for the rapidly spinning C-C 2 H 3 groups, with off-axial fluctuations of ≈ 15°. Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond, which explains its rapid photochemistry and indicates the trajectory of the 11-cis to trans isomerization.For the cryotrapped meta I state, the 2 H NMR structure showed a reduction of the polyene strain, whereas the β-ionone ring maintained its torsional twisting. Strain energy and dynamics of retinal are interpreted with regard to substituent control of receptor activation. Steric hindrance between trans retinal and Trp 265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and classical molecular mechanics simulations, and show how 2 H NMR can be applied to ligands bound to GPCRs in relation to their characteristic mechanisms of action.

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

“…Despite that three methyl environments are found for the retinal ligand in the dark state, upon 11-cis to trans isomerization ≈ two methyl sites are evident. Such a loss of retinal strain is consistent with two major planes of unsaturation following light absorption (1,34,(38)(39)(40). Application of transition state theory then allows us to connect the T 1Z results with the energetic parameters for retinal bound to rhodopsin (Table 1).…”

“…In the Meta I state, the C9-Me acts as a hinge point that prevents roll of the entire ligand. The C13-Me moves into close proximity to the β 4 strand of the second extracellular loop E2, as suggested by the 2 H NMR structure of retinal in Meta I (40). As a result of 11-cis to trans isomerization, the steric force is transmitted via the C13-Me group to the extracellular E2 loop (Fig.…”

“…The residual quadrupolar couplings hχ Q i directly yield order parameters for the rapidly spinning methyl groups (40). The principal axis system (PAS) of EFG tensor of 2 H nucleus is denoted by P (z-axis parallel to C-2 H bond); I is intermediate frame for methyl rotation (z-axis is C-C 2 H 3 bond; not shown); M is methyl coordinate system (z-axis is average C-C 2 H 3 bond direction); D is membrane system (z-axis is surface normal n 0 ), and L is the laboratory system (z-axis along external magnetic field B 0 ) (31).…”

“…In Fig. 5 we combine the 2 H NMR relaxation data with our FTIR studies (17,27) and other measurements (18,19,23,40,45) of μs-ms functional protein movements. In the Meta I state, the C9-Me acts as a hinge point that prevents roll of the entire ligand.…”

“…The β-ionone ring occupies a hydrophobic pocket that encompasses Met 207 , Phe 208 , His 211 , and Phe 212 of H5-it is another key anchor point for retinal (33). The high E a barrier for the C5-Me in Meta I (Table 1) suggests the isomerization force (strain energy) is transmitted via the β-ionone ring towards a second hydrogen-bonding network connecting helices H5 and H3 (40). Expulsion of the β-ionone ring from its hydrophobic cavity (46) or a large movement of retinal are rendered unlikely (25).…”