Structural basis for sensory rhodopsin function

The crystal structure of sensory rhodopsin II from Natronobacterium pharaonis was recently solved at 2.1 Å resolution from lipidic cubic phase-grown crystals. A critical analysis of previous structure-function studies is possible within the framework of the high-resolution structure of this photoreceptor. Based on the structure, a molecular understanding emerges of the efficiency and selectivity of the photoisomerization reaction, of the interaction of the sensory receptor and its cognate transducer protein HtrII, and of the mechanism of spectral tuning in photoreceptors. The architecture of the retinal binding pocket is compact, representing a major determinant for the selective binding of the chromophore, all-trans retinal to the apoprotein, opsin. Several chromophore-protein interactions revealed by the structure were not predicted by previous mutagenesis and spectroscopic analyses. The structure suggests likely mechanisms by which photoisomerization triggers the activation of sensory rhodopsin II, and highlights the possibility of a unified mechanism of signaling mediated by sensory receptors, including visual rhodopsins. Future investigations using time-resolved crystallography, structural dynamics, and computational studies will provide the basis to unveil the molecular mechanisms of sensory receptors-mediated transmembrane signaling.     

Dynamics of the Internal Water Molecules in Squid Rhodopsin

Understanding the mechanism of G-protein coupled receptors action is of major interest for drug design. The visual rhodopsin is the prototype structure for the family A of G-protein coupled receptors. Upon photoisomerization of the covalently bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G-protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The crystal structure of the visual rhodopsin from squid solved recently suggests that a chain of water molecules extending from the retinal toward the cytoplasmic side of the protein may play a role in the signal transduction from the all-trans retinal geometry to the activated receptor. As a first step toward understanding the role of water in rhodopsin function, we performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. The simulation indicates that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region and form a persistent hydrogen-bond network in connecting Y315 to W274 via D80.     

A transporter converted into a sensor, a phototaxis signaling mutant of bacteriorhodopsin at 3.0 Å

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 Å resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C₁₄ group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174–Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.     

Methyl-accepting protein associated with bacterial sensory rhodopsin I.

In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.     

Docking of human rhodopsin mutant (Gly90→Asp) with beta-arrestin and cyanidin 3-rutinoside to cure night blindness

MOTIVATION: Rhodopsin is a visual pigment present in rod cells of retina. It belongs to GPCR family and involves photoisomerization of 11-cis-retinal to all-trans-retinal isomers, conformational changes in rhodopsin and signal transduction cascade to generate a nerve impulse. This signaling pathway has been targeted to eliminate the effect of a mutation (Gly90→Asp) responsible for abnormal activation of G-protein without retinal conformations in the absence of light leading to congenital night blindness. A theoretical model of rhodopsin with induced mutation has been deliberated in order to find potential ligands which can offset this mutational effect. The binding interactions between the target mutated rhodopsin model and potential ligands have been predicted with the help of molecular docking. The results indicated strong functional benefits of ligands as an inhibitor and an agonist for mutated rhodopsin model. Therefore, we propose a new visual cascade model which can initiate the normal signaling of rhodopsin mutant with the help of proposed ligands and can provide a hope for vision in future.     

Early structural rearrangements in the photocycle of an integral membrane sensory receptor

Sensory rhodopsins are the primary receptors of vision in animals and phototaxis in microorganisms. Light triggers the rapid isomerization of a buried retinal chromophore, which the protein both accommodates and amplifies into the larger structural rearrangements required for signaling. We trapped an early intermediate of the photocycle of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) in 3D crystals and determined its X-ray structure to 2.3 A resolution. The observed structural rearrangements were localized near the retinal chromophore, with a key water molecule becoming disordered and the retinal's beta-ionone ring undergoing a prominent movement. Comparison with the early structural rearrangements of bacteriorhodopsin illustrates how modifications in the retinal binding pocket of pSRII allow subtle differences in the early relaxation of photoisomerized retinal.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Crystallographic analysis of the primary photochemical reaction of squid rhodopsin

Visual signal transduction is initiated by the photoisomerization of 11-cis retinal upon rhodopsin ligation. Unlike vertebrate rhodopsin, which interacts with Gt-type G-protein to stimulate the cyclic GMP signaling pathway, invertebrate rhodopsin interacts with Gq-type G-protein to stimulate a signaling pathway that is based on inositol 1,4,5-triphosphate. Since the inositol 1,4,5-triphosphate signaling pathway is utilized by mammalian nonvisual pigments and a large number of G-protein-coupled receptors, it is important to elucidate how the activation mechanism of invertebrate rhodopsin differs from that of vertebrate rhodopsin. Previous crystallographic studies of squid and bovine rhodopsins have shown that there is a profound difference in the structures of the retinal-binding pockets of these photoreceptors. Here, we report the crystal structures of all-trans bathorhodopsin (Batho; the first photoreaction intermediate) and the artificial 9-cis isorhodopsin (Iso) of squid rhodopsin. Upon the formation of Batho, the central moiety of the retinal was observed to move largely towards the cytoplasmic side, while the Schiff base and the ionone ring underwent limited movements (i.e., the all-trans retinal in Batho took on a right-handed screwed configuration). Conversely, the 9-cis retinal in Iso took on a planar configuration. Our results suggest that the light energy absorbed by squid rhodopsin is mostly converted into the distortion energy of the retinal polyene chain and surrounding residues.     

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

Structural and mechanistic insight from high resolution structures of archaeal rhodopsins

Lipidic cubic phase-grown crystals yielded high resolution structures of a number of archaeal retinal proteins, the molecular mechanisms of which are being revealed as structures of photocycle intermediates become available. The structural basis for bacteriorhodopsin's mechanism of proton pumping is discussed, revealing a well-synchronized sequence of molecular events. Comparison with the high resolution structures of the halide pump halorhodopsin, as well as with the receptor sensory rhodopsin II, illustrates how small and localized structural changes result in functional divergence. Fundamental principles of energy transduction and sensory reception in the archaeal rhodopsins, which may have relevance to other systems, are discussed.     

Methyl substituents at the 11 or 12 position of retinal profoundly and differentially affect photochemistry and signalling activity of rhodopsin

The C-11=C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11=C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10-C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 degrees C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.     

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.     

Photochemical Reaction Dynamics of the Primary Event of Vision Studied by Means of a Hybrid Molecular Simulation

The photoisomerization reaction dynamics of a retinal chromophore in the visual receptor rhodopsin was investigated by means of hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations. The photoisomerization reaction of retinal constitutes the primary step of vision and is known as one of the fastest reactions in nature. To elucidate the molecular mechanism of the high efficiency of the reaction, we carried out hybrid ab initio QM/MM MD simulations of the complete reaction process from the vertically excited state to the photoproduct via electronic transition in the entire chromophore-protein complex. An ensemble of reaction trajectories reveal that the excited-state dynamics is dynamically homogeneous and synchronous even in the presence of thermal fluctuation of the protein, giving rise to the very fast formation of the photoproduct. The synchronous nature of the reaction dynamics in rhodopsin is found to originate from weak perturbation of the protein surroundings and from dynamic regulation of volume-conserving motions of the chromophore. The simulations also provide a detailed view of time-dependent modulations of hydrogen-out-of-plane vibrations during the reaction process, and identify molecular motions underlying the experimentally observed dynamic spectral modulations.     

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.     

Transducin interactions with rhodopsin. Evidence for positive cooperative behavior

Transducin and rhodopsin belong to a family of guanine nucleotide-binding (G) protein-coupled receptor systems that provide signal transduction mechanisms resulting in numerous metabolic responses in a variety of cell types. A simple, direct binding assay has been developed to investigate the molecular interactions between transducin and rhodopsin. The binding curves generated by these studies are sigmoidal, indicating an allosteric response. The Scatchard plots of this data display an asymptotic, bell-shaped character representative of the positive cooperative behavior. A Hill coefficient, nH = 1.92, was determined and found to be in close agreement with previous kinetic studies of allosterism described for rhodopsin's catalytic mechanism (Wessling-Resnick, M., and Johnson G.L. (1987) J. Biol. Chem. 262, 3697-3705). The value for Kd app was determined to be 0.05 microM. Bmax values obtained from the binding studies suggest that oligomeric complexes of rhodopsin may be involved in interactions with transducin to form multiple high affinity binding sites for the G protein. The positive cooperative behavior demonstrated in this investigation can provide insight into the molecular basis for regulation of other G protein-coupled receptor systems.     

Interaction of Asn105 with the retinal chromophore during photoisomerization of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption spectrum with a spectral shoulder, which is highly unique for the archaeal rhodopsin family. The primary reaction of ppR is a cis-trans photoisomerization of the retinal chromophore to form the K intermediate, like the well-studied proton pump bacteriorhodopsin (BR). Recent comparative FTIR spectroscopy of the K states in ppR and BR revealed that more extended structural changes take place in ppR than in BR with respect to chromophore distortion and protein structural changes [Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y., and Kamo, N. (2001) Biochemistry 40, 9238-9246]. FTIR spectroscopy of the N105D mutant protein reported here assigns the vibrational bands at 1704 and 1700 cm(-1) as C=O stretches of Asn105 in ppR and ppR(K), respectively. A comparative investigation between ppR and BR further reveals that the structure at position 105 in ppR is similar to that of the corresponding position (Asp115) in BR; this observation is supported by the recent X-ray crystallographic structures of ppR [Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich, J. L. (2001) Science 293, 1499-1503; Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroulla, E., and Navarro, J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10131-10136]. Nevertheless, structural changes upon photoisomerization at position 105 in ppR are greater than those at position 115 in BR. As a consequence of a unique chromophore-protein interaction in ppR, extended protein structural changes accompanying retinal photoisomerization occur, and these include Asn105 which is approximately 7 A from the retinal chromophore.     

Retinal Flip in Rhodopsin Activation?

Rhodopsin is a well-characterized structural model of a G protein-coupled receptor. Photoisomerization of the covalently bound retinal triggers activation. Surprisingly, the x-ray crystal structure of the active Meta-II state has a 180° rotation about the long-axis of the retinal polyene chain. Unbiased microsecond-timescale all-atom molecular dynamics simulations show that the retinal cofactor can flip back to the orientation observed in the inactive state of rhodopsin under conditions favoring the Meta-I state. Our results provide, to our knowledge, the first evidence from molecular dynamics simulations showing how rotation of the retinal ligand within its binding pocket can occur in the activation mechanism of rhodopsin.     

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.     

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.     

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.