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Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.

Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11- cis -retinal, which undergoes photo-isomerization to the all-trans form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 Å. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment.

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

Membrane proteins, encoded by ~20% of genes in almost all organisms, including humans, are critical for cellular communication, electrical and ion balances, structural integrity of the cells and their adhesions, and other functions. Atomic-resolution structures of these proteins furnish important information for understanding their molecular organization and constitute major breakthroughs in our understanding of how they participate in physiological processes. However, obtaining structural information about these proteins has progressed slowly (1, 2), mostly because of technical difficulties in the purification and handling of integral membrane proteins. Instability of the proteins in environments lacking phospholipids, the tendency for them to aggregate and precipitate, and/or difficulties with highly heterogeneous preparations of these proteins isolated from heterologous expression systems have hindered application of standard structure determination techniques to these molecules. Among membrane proteins, G-protein-coupled receptors (GPCRs)1 are of special importance because they form one of the largest and most diverse groups of receptor proteins. More than 400 nonsensory receptors identified in the human genome are involved in the regulation of virtually all physiological processes. Drug addiction, mood control, and memory (via 5-HT6 or neuropeptide receptors) are just a short list of processes in which GPCRs are critically implicated. Another even larger group of GPCRs consist of sensory receptors involved in the fundamental process of translation of light energy (rhodopsin and cone pigments), the detection of chemoattractant molecules, or the detection of compounds stimulating the taste buds (3, 4). The activity of GPCRs comes about when binding of diffusable extracellular ligands causes them to switch from quiescent forms to an active conformation capable of interaction with hundreds of G-proteins. Their roles as extracellular ligand-binding proteins make them attractive targets for drug design. GPCRs account for ~40% of all therapeutic intervention, and major GPCR research projects are found throughout the pharmaceutical industry (5, 6). A paucity of structural data is available for GPCRs. The crystal structure of a member of the largest subgroup (I) of GPCRs, rhodopsin (7), and a ligand-binding domain of the metabotropic glutamate receptor with and without the ligand (8) have been determined recently. The data allow models, firmly based on the atomic-resolution structural information, to be further tested as to the conformational changes that these receptors undergo in going from the quiescent to the signaling state. In this article, we describe the further refinement of rhodopsin (7) and provide some clues about how the receptor could be activated by light.

Through low-temperature absorption and circular dichroism (CD) spectroscopies, and G-protein (transducin) binding experiments, we have investigated molecular properties of the meta II intermediate of iodopsin, a cone visual pigment present in chicken red-sensitive cones. The meta II intermediate of iodopsin (metaiodopsin II, lambda max = 390 nm) displayed a positive CD band at about 390 nm and a large negative CD band below 300 nm. It dissociated into all-trans-retinal and the protein moiety. A long-lived intermediate corresponding to the meta III intermediate of rhodopsin was not observed in iodopsin, under our experimental conditions. Decay of metaiodopsin II was significantly suppressed in the presence of transducin, but not in the presence of both transducin and GTP, indicating that metaiodopsin II can interact with transducin and activate it. Both metaiodopsin II and metarhodopsin II displayed a large negative CD band below 300 nm. This fact suggested that during the formation of both meta II intermediates, some aromatic amino acid residues and/or a disulfide bond are rearranged, which may be important for expression of catalytic activity for exchange of GDP to GTP on transducin. On the other hand, metaiodopsin II decayed more than 10 times faster than metarhodopsin II. This fact may be one of the reasons why cones are less photosensitive than rods.

The photobleaching process of iodopsin (a chicken red-sensitive cone visual pigment) purified in a detergent system containing CHAPS and phosphatidylcholine was investigated by means of nanosecond laser photolysis at room temperature. Excitation of iodopsin with a nanosecond laser pulse (wavelength, 560 nm; pulse width, 17 ns) resulted in the formation of at least four intermediates on the nanosecond to millisecond time scale. The earliest intermediate detected had an absorption maximum at 571 nm, which was very close to that of original iodopsin (lambda max = 567 nm), and remarkably blue-shifted as compared with that of bathoiodopsin [lambda max = 625 nm; Kandori et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8908-8912]. The intermediate, named BL-iodopsin, converted to the next intermediate, lumiiodopsin (lambda max = 535 nm), with a time constant of 130 ns. The BL intermediate had an absorption maximum just between batho- and lumiiodopsins, and an extinction coefficient comparable with these intermediates. These properties are different from those of the corresponding intermediate of rhodopsin [BL(BSI)-rhodopsin], suggesting that the binding of chloride to iodopsin, but not to rhodopsin, has an influence upon changes of the chromophore-opsin interaction in the early stage of photobleaching of iodopsin. Lumiiodopsin converted to metaiodopsin I (lambda max < 500 nm) with a time constant of 230 microseconds, and then to metaiodopsin II (lambda max = 390 nm) with a time constant of 6 ms. A thermal equilibrium between metaiodopsin I and II was established, but unlike meta intermediates of rhodopsin, they showed little temperature dependence.

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.

Recent progress in high-resolution structural study of rhodopsin has been enabled by a novel selective extraction procedure with rod photoreceptor cells. In this study, we applied the method for rapid and efficient preparation of a purified analogue pigment using bovine rod outer segment membranes with 9-cis-retinal. After complete bleaching of the membranes and subsequent regeneration with the exogenous retinal, 9-cis-rhodopsin is selectively extracted from the membranes using combination of zinc and heptylthioglucoside. The solubilized sample, even with a small amount of contaminating retinal oximes, is shown to be pure enough for three-dimensional crystallization. The X-ray diffraction from 9-cis-rhodopsin crystals was examined and the electron density map at 2.9 angstroms resolution in the chromophore region can be fitted well with the model of 9-cis-retinal Schiff base.

Photoactivation of the visual rhodopsin, a prototypical G protein-coupled receptor (GPCR), involves efficient conversion of the intrinsic inverse-agonist 11- cis -retinal to the all - trans agonist. This event leads to the rearrangement of the heptahelical transmembrane bundle, which is thought to be shared by hundreds of GPCRs. To examine this activation mechanism, we determined the x-ray crystallographic model of the photoreaction intermediate of rhodopsin, lumirhodopsin, which represents the conformational state having the nearly complete all - trans agonist form of the retinal. A difference electron density map clearly indicated that the distorted all - trans -retinal in the precedent intermediate bathorhodopsin relaxes by dislocation of the β-ionone ring in lumirhodopsin, along with significant peptide displacement in the middle of helix III, including approximately two helical turns. This local movement results in the breaking of the electrostatic interhelical restraints mediated by many of the conserved residues among rhodopsin-like GPCRs, with consequent acquisition of full activity.

G-protein-coupled receptors constitute the largest transmembrane receptor family in human. They are generally activated on binding their specific ligands at the extracellular side of membranes. The signal carried by an agonist is then transmitted to the intracellular side through a conformational change of the receptor, which becomes competent to catalyse GDP/GTP exchange in the α-subunit of heterotrimeric G-protein. Since most of the G-protein-coupled receptors (rhodopsin-like subfamily) share a set of conserved amino acid residues in the transmembrane domain, it is probable that the ligand-triggered activation process involves a common mechanism of rearrangement of the hepta-helical transmembrane bundle. For understanding the nature of this event that is not yet characterized sufficiently, X-ray crystallographic studies of rhodopsin with or without light stimulation can provide valuable information. In rhodopsin, the initial cis–trans photoisomerization of retinal chromophore triggers the structural changes of transmembrane helices. This activation process has been characterized with some spectroscopically distinct photoreaction intermediates (batho, lumi, Meta I and Meta II). With recent advances in the conditions for crystallographic experiments, the diffraction limit of the rhodopsin crystals has been substantially extended. As a result, it becomes possible to detect small structural changes evoked after photoactivation under cryogenic conditions.

A new high-resolution structure is reported for bovine rhodopsin, the visual pigment in rod photoreceptor cells. Substantial improvement of the resolution limit to 2.2 A has been achieved by new crystallization conditions, which also reduce significantly the probability of merohedral twinning in the crystals. The new structure completely resolves the polypeptide chain and provides further details of the chromophore binding site including the configuration about the C6-C7 single bond of the 11-cis-retinal Schiff base. Based on both an earlier structure and the new improved model of the protein, a theoretical study of the chromophore geometry has been carried out using combined quantum mechanics/force field molecular dynamics. The consistency between the experimental and calculated chromophore structures is found to be significantly improved for the 2.2 A model, including the angle of the negatively twisted 6-s-cis-bond. Importantly, the new crystal structure refinement reveals significant negative pre-twist of the C11-C12 double bond and this is also supported by the theoretical calculation although the latter converges to a smaller value. Bond alternation along the unsaturated chain is significant, but weaker in the calculated structure than the one obtained from the X-ray data. Other differences between the experimental and theoretical structures in the chromophore binding site are discussed with respect to the unique spectral properties and excited state reactivity of the chromophore.

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophillic-lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A280/A500 value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn2+ and Cd2+) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.

Background G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices. Methodology/Principal Findings We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands. Conclusions/Significance Systematic analysis described here would be valuable for understanding of the rearrangement of seven helices which depends on the ligand specificity and activation state of the receptors.

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane α helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11- cis -retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.