Solid-State 2H NMR spectroscopy of retinal proteins in aligned membranes

Abstract

Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.

Static solid-state 2H NMR methods in studies of protein side-chain dynamics

“…Theoretical powder-type 2 H NMR spectra for C C 2 H 3 groups undergoing axial rotation (continuous colored lines) are superimposed on the experimental spectra, with residuals below. Reprinted with permission from (52), Copyright (2007) Elsevier, license number 3971590104454.…”

“…Theoretical line shapes (continuous colored lines) assume a static uniaxial distribution of rhodopsin molecules, with rotating C–C 2 H 3 groups of retinylidene cofactor. Reprinted with permission from (52), Copyright (2007) Elsevier, license number 3971590104454.…”

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

“…Solid-state 2 H NMR [91] is complementary to solid-state 13 C NMR [22, 26, 73, 120] in that orientational restraints are provided [24], which together with distance restraints [22, 73] allow site-specific structural information to be obtained analogous to solution NMR [121, 122]. In solid-state 2 H NMR of biomembranes, the motionally averaged spectral lineshapes involve residual quadrupolar couplings (RQCs) that correspond directly to the segmental order parameters [83, 123].…”

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

“…Furthermore, it was shown that binding of retinal involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH 3 -16 and CH 3 -17 [100]. Additional evidences of the interactions between retinal and rhodopsin in the ground and Meta I states have also been obtained through NMR experiments conducted variously labeling the ligand with 2 H [101–104]. These data support the hypothesis that a strain in the polyen around the cis bond assists the photoisomerization of retinal.…”