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.     

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.     

Photolysis intermediates of the artificial visual pigment cis-5,6-dihydro-isorhodopsin.

The photolysis intermediates of an artificial bovine rhodopsin pigment, cis-5,6-dihydro-isorhodopsin (cis-5,6,-diH-ISORHO, lambda max 461 nm), which contains a cis-5,6-dihydro-9-cis-retinal chromophore, are investigated by room temperature, nanosecond laser photolysis, and low temperature irradiation studies. The observations are discussed both in terms of low temperature experiments of Yoshizawa and co-workers on trans-5,6-diH-ISORHO (Yoshizawa, T., Y. Shichida, and S. Matuoka. 1984. Vision Res. 24: 1455-1463), and in relation to the photolysis intermediates of native bovine rhodopsin (RHO). It is suggested that in 5,6-diH-ISORHO, a primary bathorhodopsin intermediate analogous to the bathorhodopsin intermediate (BATHO) of the native pigment, rapidly converts to a blue-shifted intermediate (BSI, lambda max 430 nm) which is not observed after photolysis of native rhodopsin. The analogs from lumirhodopsin (LUMI) to meta-II rhodopsin (META-II) are generated subsequent to BSI, similar to their generation from BATHO in the native pigment. It is proposed that the retinal chromophore in the bathorhodopsin stage of 5,6-diH-ISORHO is relieved of strain induced by the primary cis to trans isomerization by undergoing a geometrical rearrangement of the retinal. Such a rearrangement, which leads to BSI, would not take place so rapidly in the native pigment due to ring-protein interactions. In the native pigment, the strain in BATHO would be relieved only on a longer time scale, via a process with a rate determined by protein relaxation.     

Coupling of retinal, protein, and water dynamics in squid rhodopsin

The light-induced isomerization of the retinal from 11-cis to all-trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11-cis and all-trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment.     

Transition dipole orientations in the early photolysis intermediates of rhodopsin.

The linear dichroism spectrum of rhodopsin in sonicated bovine disk membranes was measured 30, 60, 170, and 600 ns after room temperature photolysis with a linearly polarized, 7-ns laser pulse (lambda = 355 or 477 nm). A global exponential fitting procedure based on singular value decomposition was used to fit the linear dichroism data to two exponential processes which differed spectrally from one another and whose lifetimes were 42 +/- 7 ns and 225 +/- 40 ns. These results are interpreted in terms of a sequential model where bathorhodopsin (BATHO, lambda max = 543 nm) decays toward equilibrium with a blue shifted intermediate (BSI, lambda max = 478 nm). BSI then decays to lumirhodopsin (LUMI, lambda max = 492 nm). It has been suggested that two bathorhodopsins decay in parallel to their products. However, a Monte Carlo simulation of partial photolysis of solid-state visual pigment samples shows that one mechanism which creates populations of BATHO having different photolysis rates at 77 K may not be responsible for the two decay rates reported here at room temperature. The angle between the cis band and 498-nm band transition dipoles of rhodopsin is determined to be 38 degrees. The angles between both these transition dipoles and those of the long-wave-length bands of BATHO, BSI, and LUMI are also determined. It is shown that when BATHO is formed its transition dipole moves away from the original cis band transition dipole direction. The transition dipole then moves roughly twice as much towards the original cis band direction when BSI appears. Production of LUMI is associated with return of the transition dipole almost to the original orientation relative to the cis band, but with some displacement normal to the plane which contains the previous motions. The correlation between the lambda max of an intermediate and its transition dipole direction is discussed.     

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Binding of more than one retinoid to visual opsins

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.     

Chimeric microbial rhodopsins containing the third cytoplasmic loop of bovine rhodopsin

G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000–140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.     

Nanosecond photolysis of rhodopsin: evidence for a new, blue-shifted intermediate

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.     

The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.     

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is ∼20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.Rhodopsin, the photosensitive G protein-coupled receptor (GPCR),³ is responsible for transmitting a light signal into an intracellular signaling cascade through activation of G protein in visual and non-visual photoreceptor cells. Rhodopsin consists of a protein moiety (opsin, comprising seven transmembrane α-helical segments) combined with a chromophore (11-cis retinal) that acts as the light-sensitive ligand. Photoisomerization of the 11-cis retinal to the all-trans form induces structural changes in the protein moiety that then enable it to couple with and activate the G protein.The crystal structure of inactive bovine rhodopsin has been extensively investigated (1–3). Recently, a crystal structure of inactive invertebrate squid rhodopsin was also solved (4), and crystal structures of the inactive form of β-adrenergic receptors and A2 adenosine receptor have been reported (5–7). Remarkably, all of these crystal structures exhibit a very similar arrangement for the seven transmembrane helices (4, 8). Together, these facts suggest that the architecture for the inactive form is conserved among rhodopsin-like GPCRs.The structural features of an activated GPCR are much less defined. Thus, a variety of biochemical and biophysical methods, including cross-linking methods (9, 10) and site-directed spin and fluorescence labeling methods (10–13), have been employed to identify the dynamic and structural changes involved in forming the activated state. The data from these studies consistently suggest that some kind of movement of helix VI is involved in the formation of the active state of the rhodopsins. In particular, the cytoplasmic end of helix VI has been proposed to rotate and/or tilt toward helix V (10–13). Remarkably, the recent crystal structures of bovine opsin are consistent with the widely accepted helix motion model. Both the structures of opsin (the ligand-free form of rhodopsin that has partial G protein activation ability) and a complex of opsin with a peptide derived from the G protein C terminus show a movement of helix VI toward helix V, compared with the dark state rhodopsin structure (14, 15). Studies of β-adrenergic and muscarinic receptors also show that agonist binding promotes movement of helix VI toward helix V in these receptors (16, 17). Because the region between the cytoplasmic ends of helices V and VI in various GPCRs is a main site of interaction with G proteins (18), it is possible that movement of helices V and VI leads to formation of a conformation capable of interacting with G protein (19).Together, these studies imply that the active state conformation of GPCRs may be similar. However, a detailed comparison of the active-state conformation for two different GPCRs has never been precisely undertaken in the same laboratory using the same methods.In this context we have been investigating rhodopsins with different functional properties to determine whether their active states have different conformations. Our goal was to determine whether any functional or structural differences in the active states of these GPCRs could be detected under the exact same experimental conditions.Previously, we have found that several rhodopsins, such as an invertebrate rhodopsin and a vertebrate non-visual rhodopsin parapinopsin (20, 21), can be activated not only by light but also by exogenous all-trans retinal acting as a full agonist (22). This is in contrast to vertebrate visual rhodopsins, including bovine rhodopsin, which cannot fully form the active state by direct binding of all-trans retinal (23), although all-trans retinal can fully activate some rhodopsin mutants (24). Other invertebrate rhodopsin (25) and the circadian photoreceptor melanopsin (26) can also bind all-trans retinal directly.Interestingly, the active form of the all-trans retinal-activated rhodopsins exhibit some striking differences in their spectroscopic and biochemical properties compared with vertebrate visual rhodopsins (27). In particular, the efficiency of bovine rhodopsin for activating G protein is ∼20∼50-fold higher than that of parapinopsin and invertebrate rhodopsin. This difference could be related to the difference in position of a specific amino acid residue counterion that is essential for rhodopsin to absorb visible light, namely one at position 113 or 181 (28).⁴ Further biochemical analyses using chimeric mutants combining rhodopsins with lower and higher G protein activation abilities suggested that the difference in G protein activation ability was because of a structural difference in transmembrane helices in the active states but not because of difference in amino acid sequence of G protein interaction site (29) (Fig. 1, A–C). In addition, the active states of parapinopsin and the invertebrate rhodopsin are thermally stable and can be reconverted to the inactive state by subsequent light absorption, showing photo-regenerable or bistable nature (21, 28), unlike the active state of bovine rhodopsin, which is thermally unstable and cannot revert to the inactive state by subsequent light absorption (30).Open in a separate windowFIGURE 1.Molecular properties and sites of fluorescent probe attachment for bovine rhodopsin and parapinopsin. A, sequence alignment of bovine rhodopsin and parapinopsin. Amino acid residues to which cysteine and fluorescence label were introduced are marked with red. The amino acid residues identical and similar between bovine rhodopsin and parapinopsin are shown with white characters with black and gray background, respectively. Bovine rhodopsin and parapinopsin show 41% sequence identity and 61% similarity. In this paper the residue number of parapinopsin is described by using the bovine rhodopsin numbering system. B and C, comparison of G protein activation ability of rhodopsin and parapinopsin wild type (WT) proteins and loop-replaced mutants. In these mutants the second and/or third cytoplasmic loop was swapped between the two receptors. ParaL2 and ParaL3 indicate mutants of bovine rhodopsin in which second and third loops were replaced with the corresponding loop of parapinopsin, respectively. RhoL2 and RhoL3 indicate mutants of parapinopsin in which the second and third loops were replaced with the corresponding loops of bovine rhodopsin, respectively. ParaL2L3 and RhoL2L3 are mutants of bovine rhodopsin and parapinopsin in which both the second and third loops were swapped, respectively. See Terakita et al. (29) for more details. Data are presented as the means ± S.E. of three separate experiments except for mutants RhoL3, RhoL2L3, and ParaL2L3 (n = 2). D, model of bovine rhodopsin. Amino acid residues which were mutated to cysteine to enable attachment of the fluorescent probe bimane or mutated to tryptophan are indicated. Positions 226, 227, 244, 250, and 251 in the crystal structure of the dark state of bovine rhodopsin (PDB code 1GZM) are shown. E, reaction of the mBBr label with a sulfhydryl group. The mutants labeled with mBBr are named by the number of the residue and the suffix B₁. F, reaction of the PDT-bimane with a sulfhydryl group. The mutants labeled with PDT-bimane are named by the number of the residue and the suffix B₂. The disulfide linkage between the label and protein can be cleaved using Tris(2-carboxyethyl)phosphine (32).In this study we used site-directed fluorescence labeling (13, 31) to compare the structural features of active states of bovine rhodopsin with lamprey parapinopsin, a UV-sensitive non-visual pigment in the pineal organs (21). Parapinopsin shows relatively high sequence similarity (∼60%) to bovine rhodopsin, yet it has a greatly reduced ability to activate G protein (see Fig. 1, A–C) (21, 28). Using established protocols, we introduced cysteine residues into the cytoplasmic ends of helices V and VI, the region proposed to rearrange upon activation in GPCRs (11, 12, 14, 18). We then site-specifically labeled these cysteines with the small, non-polar fluorescent probe, bimane, and used the spectral properties of these bimane probes to act as reporter groups for environmental changes around their site of attachment upon formation of the photoactivated state for both rhodopsins.In addition, we measured changes in the relative proximity of the cytoplasmic ends of helix VI to helix V in both rhodopsin and parapinopsin using the tryptophan-induced-quenching of bimane (TrIQ-bimane) fluorescence method (31, 32). TrIQ-bimane measures the efficiency of intramolecular fluorescence quenching of bimane caused by tryptophan (Trp), which occurs in a distance-dependent manner. The goal of this study was to determine whether the helices in both receptors moved in the same way during formation of the active state. Our results show that whereas movement of helix VI relative to helix V occurs during formation of the active state for both parapinopsin and bovine rhodopsin, the “amplitude” of the movement is markedly different between the two rhodopsins.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

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.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Renewal of visual pigment in photoreceptors of the blowfly

Summary Spectrophotometric measurements of photoreceptors 1–6 in the blowfly demonstrate that rhodopsin undergoes a continuous renewal. This involves, in the dark, the slow degradation of rhodopsin whereas metarhodopsin is degraded at a much faster rate. The effect of light is to reduce the rate at which metarhodopsin is degraded, i.e. the rate is inversely related to the intensity of the light. Rhodopsin synthesis is dependent on the presence of 11-cis retinal which is formed via a photoreaction from all-trans retinal resulting from the breakdown of rhodopsin and/or metarhodopsin: the biosynthesis of rhodopsin is therefore a light dependent process. Light of the blue/violet spectral range was found to mediate the isomerization of all-trans retinal into the 11-cis form. It is proposed that this stereospecificity is the result of all-trans retinal being bound to a protein. On the basis of the results a visual pigment cycle is proposed.     

Excited-state dynamics of all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

The rhodopsin mimic is a chemically synthetized complex with retinyl Schiff base (RSB) formed between protein and the retinal chromophore that can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the ultrafast transient absorption spectra of three mutants of the cellular retinoic acid-binding protein II-based rhodopsin mimic at acidic environment were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the β-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. The experimental results provide implications on designing a rhodopsin-like protein with significant infrared fluorescence, which can be particularly useful in the applications in biosensing or bioimaging in deeper tissues.     

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.     

Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.     

Using Total Internal Reflection Fluorescence Microscopy To Visualize Rhodopsin-Containing Cells

Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low—comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls—these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.