Hydration-mediated G-protein-coupled receptor activation.

The Rhodopsin family of G-protein­coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80­100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.

Activation of the G-Protein-Coupled Receptor Rhodopsin by Water.

Visual rhodopsin is an important archetype for G-protein-coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent-swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force-based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin-II state. Moreover, we discovered that binding and release of the C-terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration-dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G-protein cycle.

Quantum Mechanical and Molecular Mechanics Modeling of Membrane-Embedded Rhodopsins.

Computational chemistry provides versatile methods for studying the properties and functioning of biological systems at different levels of precision and at different time scales. The aim of this article is to review the computational methodologies that are applicable to rhodopsins as archetypes for photoactive membrane proteins that are of great importance both in nature and in modern technologies. For each class of computational techniques, from methods that use quantum mechanics for simulating rhodopsin photophysics to less-accurate coarse-grained methodologies used for long-scale protein dynamics, we consider possible applications and the main directions for improvement.

Synthesis of 9-CD3-9-cis-Retinal Cofactor of Isorhodopsin.

We report the synthesis of 9-CD3-9-cis-retinal via a six-step procedure from ß-ionone. The steps involve an initial deuteration of the methyl ketone of ß-ionone followed by two consecutive Horner-Wadsworth-Emmons (HWE) coupling reactions and their corresponding DIBAL reductions. A final oxidation of the allylic alcohol of the retinol leads to the target compound. This deuterium labeled retinoid is an important cofactor for studying protein-retinoid interactions in isorhodopsin.

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

Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state (2)H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific (2)H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state (2)H NMR relaxation (spin-lattice, T(1Z), and quadrupolar-order, T(1Q)) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation--even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the (2)H NMR data suggest the ß-ionone ring remains in its hydrophobic binding pocket in all three states of the protein. We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the ß-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response--rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.

Osmotic stress studies of G-protein-coupled receptor rhodopsin activation.

We summarize and critically review osmotic stress studies of the G-protein-coupled receptor rhodopsin. Although small amounts of structural water are present in these receptors, the effect of bulk water on their function remains uncertain. Studies of the influences of osmotic stress on the GPCR archetype rhodopsin have given insights into the functional role of water in receptor activation. Experimental work has discovered that osmolytes shift the metarhodopsin equilibrium after photoactivation, either to the active or inactive conformations according to their molar mass. At least 80 water molecules are found to enter rhodopsin in the transition to the photoreceptor active state. We infer that this movement of water is both necessary and sufficient for receptor activation. If the water influx is prevented, e.g., by large polymer osmolytes or by dehydration, then the receptor functional transition is back shifted. These findings imply a new paradigm in which rhodopsin becomes solvent swollen in the activation mechanism. Water thus acts as an allosteric modulator of function for rhodopsin-like receptors in lipid membranes.

Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation.

Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.

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

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 beta-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 beta4 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.

Phase separation in binary mixtures of bipolar and monopolar lipid dispersions revealed by 2H NMR spectroscopy, small angle x-ray scattering, and molecular theory.

Binary mixtures of C(20)BAS and POPC membranes were studied by solid-state (2)H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C(20)BAS derivatives--[1',1',20',20'-(2)H(4)]C(20)BAS, [2',2',19',19'-(2)H(4)]C(20)BAS, and [10',11'-(2)H(2)]C(20)BAS--combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as membranes containing POPC-d(31) and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The (2)H NMR spectra of [10',11'-(2)H(2)]C(20)BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At < or =50 mol % C(20)BAS, smaller quadrupolar couplings appear in the spectra, indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids increases as the amount of POPC in the membrane increases; however, the transmembrane component remains the dominant bolalipid conformation in the membrane even at 45 degrees C and 10 mol % C(20)BAS, where it accounts for approximately 50% of the bolalipid population. The large fraction of C(20)BAS transmembrane conformers, regardless of the C(20)BAS/POPC ratio, together with the findings from molecular mean-field theory calculations, suggests the coexistence of phase-separated bolalipid-rich domains and POPC-rich domains. A single lamellar repeat distance was observed in SAXS experiments corresponding to the average repeat spacing expected for C(20)BAS- and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C(20)BAS hydrophobic mismatch.

Retinal conformation and dynamics in activation of rhodopsin illuminated by solid-state H NMR spectroscopy.

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 SC(3) approximately 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 beta-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 beta-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the beta-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.

Fluid mechanical matching of H+-ATP synthase subunit c-ring with lipid membranes revealed by 2H solid-state NMR.

The F(1)F(o)-ATP synthase utilizes the transmembrane H(+) gradient for the synthesis of ATP. F(o) subunit c-ring plays a key role in transporting H(+) through F(o) in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d(54)) at lipid/protein ratios of 50:1 and 20:1 by means of (2)H-solid-state NMR. In the liquid-crystalline state of DMPC, the (2)H-NMR moment values and the order parameter (S(CD)) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the (2)H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F(1)F(o)-ATPse molecular motor.

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

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.

Bolalipid membrane structure revealed by solid-state 2H NMR spectroscopy.

Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1',1',20',20'-2H4]C20BAS-PC, [2',2',19',19'-2H4]C20BAS-PC, or [10',11'-2H2]C20BAS-PC, were analyzed by 2H NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C20BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 A and 60.4 A2, respectively, at 25 degrees C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC.

Structural analysis and dynamics of retinal chromophore in dark and meta I states of rhodopsin from 2H NMR of aligned membranes.

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.

Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation.

Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared with alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure, rhodopsin is swollen by the penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.

Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy.

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state (2)H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover (2)H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state (2)H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition we review optimized conditions for trapping the rhodopsin photointermediates; and we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films.

Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation.

X-ray and magnetic resonance approaches, though central to studies of G protein-coupled receptor (GPCR)-mediated signaling, cannot address GPCR protein dynamics or plasticity. Here we show that solid-state (2)H NMR relaxation elucidates picosecond-to-nanosecond-timescale motions of the retinal ligand that influence larger-scale functional dynamics of rhodopsin in membranes. We propose a multiscale activation mechanism whereby retinal initiates collective helix fluctuations in the meta I-meta II equilibrium on the microsecond-to-millisecond timescale.

Solid-state 2H NMR structure of retinal in metarhodopsin I.

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 beta-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.

Deuterium NMR structure of retinal in the ground state of rhodopsin.

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, 7636-7643] 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 beta-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.