Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR (original) (raw)
Related papers
Journal of Molecular Biology, 2007
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 darkstate 2 H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the β-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of ≈ 0.9 for the 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 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 β-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.
Solid-State 2H NMR spectroscopy of retinal proteins in aligned membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
Solid-state 2 H 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. 2 H 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-C 2 H 3 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 2 H 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 β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2 H 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 2 H NMR is thus illuminating in terms of their different biological functions.
Photochemistry and Photobiology, 2009
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. †This paper is part of the
Deuterium NMR Structure of Retinal in the Ground State of Rhodopsin †
Biochemistry, 2004
The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ( 2 H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal 2 H-labeled at the C 5 , C 9 , or C 13 methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of 2 H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121,[7636][7637][7638][7639][7640][7641][7642][7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the -ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state 2 H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations. mean square deviation; MNDO, modified neglect of diatomic overlap; TD, transition dipole.
Structure and dynamics of retinal in rhodopsin elucidated by deuterium solid state NMR
2004
Rhodopsin is a seven transmembrane helix GPCR found which mediates dim light vision, in which the binding pocket is occupied by the ligand 11- cis-retinal. A site-directed 2H-labeling approach utilizing solid-state 2H NMR spectroscopy was used to investigate the structure and dynamics of retinal within its binding pocket in the dark state of rhodopsin, and as well the MetaI and
Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state 2 H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific 2 H labels have been introduced into the methyl groups of retinal and solid-state 2 H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent 2 H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu 122 and Trp 265 within the binding pocket. The solid-state 2 H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.
Solid-State 2 H NMR Structure of Retinal in Metarhodopsin I
Journal of the American Chemical Society, 2006
The structural and photochemical changes in rhodopsin due to absorption of light are crucial for understanding the process of visual signaling. We investigated the structure of trans-retinal in the metarhodopsin I photointermediate (MI), where the retinylidene cofactor functions as an antagonist. Rhodopsin was regenerated using retinal that was 2 H-labeled at the C5, C9, or C13 methyl groups and was reconstituted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Membranes were aligned by isopotential centrifugation, and rhodopsin in the supported bilayers was then bleached and cryotrapped in the MI state. Solid-state 2 H NMR spectra of oriented rhodopsin in the low-temperature lipid gel state were analyzed in terms of a static uniaxial distribution (Nevzorov, A. A.; Moltke, S.; Heyn, M. P.; Brown, M. F. J. Am. Chem. Soc. 1999, 121, 7636-7643). The line shape analysis allowed us to obtain the methyl bond orientations relative to the membrane normal in the presence of substantial alignment disorder (mosaic spread). Relative orientations of the methyl groups were used to calculate effective torsional angles between the three different planes that represent the polyene chain and the -ionone ring of retinal. Assuming a three-plane model, a less distorted structure was found for retinal in MI compared to the dark state. Our results are pertinent to how photonic energy is channeled within the protein to allow the strained retinal conformation to relax, thereby forming the activated state of the receptor.
NMR studies of retinal proteins
Journal of Bioenergetics and Biomembranes, 1992
A review is given of the use of nuclear magnetic resonance (NMR) spectroscopy to study bacteriorhodopsin and bovine rhodopsin. Solution and solid-state approaches are included. The studies of the bacterial proton pump examine the chromophore, the peptide backbone, and the protein side chains. The studies of the bovine visual pigment are limited to the chromophore. Various forms of each pigment are considered. Both structural and dynamic features are addressed.
Solid-State Deuterium NMR Spectroscopy of Rhodopsin
Rhodopsin is a prototype for the large Family A of G-protein–coupled receptors (GPCRs). These proteins regulate many signaling processes, and more than 35% of human pharmaceuticals are targeted against diseases related to dysfunctions of GPCR pathways. Membrane proteins such as GPCRs are challenging to crystallize for X-ray studies. In addition, their effective molar masses in detergent solutions push the limits for solution NMR spectroscopy. By contrast, solid-state NMR allows both the structure and dynamics of membrane proteins to be investigated in a natural lipid bilayer environment. Here, we describe solid-state 2 H NMR methods for investigating structural and dynamical changes of the retinylidene cofactor of the GPCR rhodopsin upon photoillumination. Rhodopsin was regenerated with retinal containing 2 H-labeled C5-, C9-, and C13-methyl groups. The receptor was recombined with phospholipid membranes, which were aligned on planar glass slides. The angular dependences of the 2 H NMR spectra and the corresponding relaxation rates were measured for rhodopsin in the dark and in the cryo-trapped preactive Meta-I and active Meta-II states. Analysis of the 2 H NMR lineshapes using a static uniaxial distribution yields orientational restraints for the retinylidene conformation when bound to the protein. Solid-state 2 H NMR relaxation data provide additional information on the motion of the bound cofactor. The structural and dynamical changes of retinal reveal how its functional groups (methyl groups and the β-ionone ring) affect rhodopsin light