Rhodopsin 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 low-temperature 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 plus the alignment disorder (mosaic spread). The dark-state (2)H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the beta-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of approximately 0.9 for the spinning C-C(2)H(3) groups, with off-axial fluctuations of approximately 15 degrees . Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond that explains its rapid photochemistry and the trajectory of 11-cis to trans isomerization. In the cryo-trapped meta I state, the (2)H NMR structure shows a reduction of the polyene strain, while torsional twisting of the beta-ionone ring is maintained. Distortion of the retinal conformation is interpreted through substituent control of receptor activation. Steric hindrance between trans retinal and Trp265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and molecular mechanics simulations of ligands bound to GPCRs, and illustrate how (2)H NMR can be applied to study their biological mechanisms of action.
Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the beta-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the beta-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The beta-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state (2)H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.
Abstract:
The dynamics, structure, and thermotropic behavior of a new class of lipophilic [60]fullerene (C60) derivatives, so-called lipo-fullerenes, have been studied by differential scanning calorimetry (DSC), deuterium nuclear magnetic resonance (2H NMR), and X-ray scattering. The lipo-fullerene studied consists of six pairs of perdeuterated C18 alkyl chains as substituents of six covalently attached methylene groups in octahedral sites. The symmetry of this highly symmetrical hexamethanofullerene is Th We find drastic changes of the molecular arrangement of the lipo-fullerenes induced by temperature. Heating the sample from 20 to 70 °C causes it to undergo two major structural transitions. At 55 °C we observe an exothermic transition from a low-temperature, hard sphere-like packing state of the molecules, with separation distances (6.1 nm) slightly above the maximum diameter of the molecules, to a condensed one. This latter state involves partial intercalation (interdigitation) of the alkyl chains belonging to adjacent molecules and is preceded by partial melting of the chains to accommodate sterically for the (exothermic) interdigitation. The latter allows denser packing with an average separation distance of 4.8 nm. At a temperature of 64 °C, an endothermic melting transition from the interdigitated to a viscous fluidlike state is observed, with an average separation distance of 2.8 nm. Cooling the sample from 70 °C causes a direct transition from the fluid into the low-temperature state with no interdigitation of the chains.
Abstract:
Material properties of lipid bilayers were studied on the mesoscopic scale using deuterium nuclear magnetic resonance spectroscopy. The fluid phase of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was compared with DMPC containing a nonionic detergent as an additive. Order parameter profiles were obtained from the deuterium NMR spectra of DMPC having perdeuterated acyl chains (DMPC-J54). A reduction of the order parameters of DMPC-d54 in the presence of the detergent octaethyleneglycol-mono-/i-dodecyl ether (C12E8) was observed, consistent with an increased configurational freedom of the phospholipid acyl chains. Relaxation rates R1z(i) and R1Q(i) were measured and spectral densities Jm(mω0) where in = 1,2 were calculated from the combined relaxation results. Profiles of the observables, i.e., order parameters, relaxation rates, and spectral densities were interpreted within the framework of a new composite membrane deformation model, which describes characteristic properties of the membrane in terms of a continuum picture. According to this model, the influence of the nonionic detergent (C12E8) on the electroneutral DMPC membrane is to increase the membrane flexibility as manifested by the functional dependence of the R1z(i) and R1Q(i) rates, i.e., the dependence of the spectral densities on the corresponding profiles of the orientational order parameters |SCD(i)|. Within the theoretical framework the increased flexibility of the detergent-containing membranes corresponds to a decrease of the elastic constants for continuum (elastic) deformations of the membrane bilayer. In the case of splay fluctuations the elastic constant and the bilayer thickness are related to the macroscopic bending rigidity, which qualitatively yields a correspondence to studies of macroscopic bending fluctuations thus yielding support for the model. In general, these findings indicate a softening of the membrane bilayer by the presence of a nonionic detergent, which corresponds to a decrease of the elastic constants for continuum deformations of the membrane. © 2000 American Chemical Society.
