Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Chromophore Structure in an Inactive State of a Novel Photosensor Protein Opn5L1: Resonance Raman Evidence for the Formation of a Deprotonated Adduct at the 11th Carbon Atom.

Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Role of Monomer/Tetramer Equilibrium of Rod Visual Arrestin in the Interaction with Phosphorylated Rhodopsin.

The phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation.

Creation of photocyclic vertebrate rhodopsin by single amino acid substitution.

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Amino acid residue at position 188 determines the UV-sensitive bistable property of vertebrate non-visual opsin Opn5.

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.

Evolutionary adaptation of visual pigments in geckos for their photic environment.

Vertebrates generally have a single type of rod for scotopic vision and multiple types of cones for photopic vision. Noteworthily, nocturnal geckos transmuted ancestral photoreceptor cells into rods containing not rhodopsin but cone pigments, and, subsequently, diurnal geckos retransmuted these rods into cones containing cone pigments. High sensitivity of scotopic vision is underlain by the rod's low background noise, which originated from a much lower spontaneous activation rate of rhodopsin than of cone pigments. Here, we revealed that nocturnal gecko cone pigments decreased their spontaneous activation rates to mimic rhodopsin, whereas diurnal gecko cone pigments recovered high rates similar to those of typical cone pigments. We also identified amino acid residues responsible for the alterations of the spontaneous activation rates. Therefore, we concluded that the switch between diurnality and nocturnality in geckos required not only morphological transmutation of photoreceptors but also adjustment of the spontaneous activation rates of visual pigments.

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

Session 2SFA-the symposium "Elucidation of biological functions by optical control" on BSJ2019 at Miyazaki, Japan.

The symposium "Elucidation of biological functions by optical control" was held during the 57th annual meeting of the Biophysical Society of Japan (BSJ2019) at Miyazaki, Japan. In this commentary, we introduce invited speakers of this symposium and summarized their research topics.

Conformational Differences among Metarhodopsin I, Metarhodopsin II, and Opsin Probed by Wide-Angle X-ray Scattering.

Among the photoproducts of vertebrate rhodopsin, only metarhodopsin II (Meta-II) preferentially adopts the active structure in which transmembrane helices are rearranged. Light-induced helical rearrangement of rhodopsin in membrane-embedded form was directly monitored by wide-angle X-ray scattering (WAXS) using nanodiscs. The change in the WAXS curve for the formation of Meta-II was characterized by a peak at 0.2 Å-1 and a valley at 0.6 Å-1, which were not observed in metarhodopsin I and opsin. However, acid-induced active opsin (Opsin*) showed a 0.2 Å-1 peak, but no 0.6 Å-1 valley. Analyses using the model structures based on the crystal structures of dark state and Meta-II suggest that the outward movement of helix VI occurred in Opsin*. However, the displaced helices III and V in Meta-II resulting from the disruption of cytoplasmic ionic lock were restored in Opsin*, which is likely to destabilize the G-protein-activating structure of opsin.

Evolutionary history of teleost intron-containing and intron-less rhodopsin genes.

Recent progress in whole genome sequencing has revealed that animals have various kinds of opsin genes for photoreception. Among them, most opsin genes have introns in their coding regions. However, it has been known for a long time that teleost retinas express intron-less rhodopsin genes, which are presumed to have been formed by retroduplication from an ancestral intron-containing rhodopsin gene. In addition, teleosts have an intron-containing rhodopsin gene (exo-rhodopsin) exclusively for pineal photoreception. In this study, to unravel the evolutionary origin of the two teleost rhodopsin genes, we analyzed the rhodopsin genes of non-teleost fishes in the Actinopterygii. The phylogenetic analysis of full-length sequences of bichir, sturgeon and gar rhodopsins revealed that retroduplication of the rhodopsin gene occurred after branching of the bichir lineage. In addition, analysis of the tissue distribution and the molecular properties of bichir, sturgeon and gar rhodopsins showed that the abundant and exclusive expression of intron-containing rhodopsin in the pineal gland and the short lifetime of its meta II intermediate, which leads to optimization for pineal photoreception, were achieved after branching of the gar lineage. Based on these results, we propose a stepwise evolutionary model of teleost intron-containing and intron-less rhodopsin genes.

Elementary response triggered by transducin in retinal rods.

G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps-/- background, gives an effective gain of only ∼12-14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.

Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates.

Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.

Single-molecule diffusion-based estimation of ligand effects on G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are major drug targets. Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity. We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs. Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions. Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively. Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits. We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein-coupling selectivity.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Genotype determination of the OPN1LW/OPN1MW genes: novel disease-causing mechanisms in Japanese patients with blue cone monochromacy.

Blue cone monochromacy (BCM) is characterized by loss of function of both OPN1LW (the first) and OPN1MW (the downstream) genes on the X chromosome. The purpose of this study was to investigate the first and downstream genes in the OPN1LW/OPN1MW array in four unrelated Japanese males with BCM. In Case 1, only one gene was present. Abnormalities were found in the promoter, which had a mixed unique profile of first and downstream gene promoters and a -71A > C substitution. As the promoter was active in the reporter assay, the cause of BCM remains unclear. In Case 2, the same novel mutation, M273K, was present in exon 5 of both genes in a two-gene array. The mutant pigments showed no absorbance at any of the wavelengths tested, suggesting that the mutation causes pigment dysfunction. Case 3 had a large deletion including the locus control region and entire first gene. Case 4 also had a large deletion involving exons 2-6 of the first gene. As an intact LCR was present upstream and one apparently normal downstream gene was present, BCM in Case 4 was not ascribed solely to the deletion. The deletions in Cases 3 and 4 were considered to have been caused by non-homologous recombination.

Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein-Coupled Receptor, Rhodopsin.

Constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retinas, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type, but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Single-molecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift toward the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics.

Opn5L1 is a retinal receptor that behaves as a reverse and self-regenerating photoreceptor.

Most opsins are G protein-coupled receptors that utilize retinal both as a ligand and as a chromophore. Opsins' main established mechanism is light-triggered activation through retinal 11-cis-to-all-trans photoisomerization. Here we report a vertebrate non-visual opsin that functions as a Gi-coupled retinal receptor that is deactivated by light and can thermally self-regenerate. This opsin, Opn5L1, binds exclusively to all-trans-retinal. More interestingly, the light-induced deactivation through retinal trans-to-cis isomerization is followed by formation of a covalent adduct between retinal and a nearby cysteine, which breaks the retinal-conjugated double bond system, probably at the C11 position, resulting in thermal re-isomerization to all-trans-retinal. Thus, Opn5L1 acts as a reverse photoreceptor. We conclude that, like vertebrate rhodopsin, Opn5L1 is a unidirectional optical switch optimized from an ancestral bidirectional optical switch, such as invertebrate rhodopsin, to increase the S/N ratio of the signal transduction, although the direction of optimization is opposite to that of vertebrate rhodopsin.

Drosophila melanogaster rhodopsin Rh7 is a UV-to-visible light sensor with an extraordinarily broad absorption spectrum.

The genome of Drosophila melanogaster contains seven rhodopsin genes. Rh1-6 proteins are known to have respective absorption spectra and function as visual pigments in ocelli and compound eyes. In contrast, Rh7 protein was recently revealed to function as a circadian photoreceptor in the brain. However, its molecular properties have not been characterized yet. Here we successfully prepared a recombinant protein of Drosophila Rh7 in mammalian cultured cells. Drosophila Rh7 bound both 11-cis-retinal and 11-cis-3-hydroxyretinal to form photo-pigments which can absorb UV light. Irradiation with UV light caused formation of a visible-light absorbing metarhodopsin that activated Gq-type of G protein. This state could be photoconverted back to the original state and, thus Rh7 is a Gq-coupled bistable pigment. Interestingly, Rh7 (lambda max = 350 nm) exhibited an unusual broad spectrum with a longer wavelength tail reaching 500 nm, whose shape is like a composite of spectra of two pigments. In contrast, replacement of lysine at position 90 with glutamic acid caused the formation of a normal-shaped absorption spectrum with maximum at 450 nm. Therefore, Rh7 is a unique photo-sensor that can cover a wide wavelength region by a single pigment to contribute to non-visual photoreception.

Evolutionary steps involving counterion displacement in a tunicate opsin.

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate- and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MI-like and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate- and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.