Cone visual pigments

Cone visual pigments are visual opsins that are present in vertebrate cone photoreceptor cells and act as photoreceptor molecules responsible for photopic vision. Like the rod visual pigment rhodopsin, which is responsible for scotopic vision, cone visual pigments contain the chromophore 11-cis-retinal, which undergoes cis–trans isomerization resulting in the induction of conformational changes of the protein moiety to form a G protein-activating state. There are multiple types of cone visual pigments with different absorption maxima, which are the molecular basis of color discrimination in animals. Cone visual pigments form a phylogenetic sister group with non-visual opsin groups such as pinopsin, VA opsin, parapinopsin and parietopsin groups. Cone visual pigments diverged into four groups with different absorption maxima, and the rhodopsin group diverged from one of the four groups of cone visual pigments. The photochemical behavior of cone visual pigments is similar to that of pinopsin but considerably different from those of other non-visual opsins. G protein activation efficiency of cone visual pigments is also comparable to that of pinopsin but higher than that of the other non-visual opsins. Recent measurements with sufficient time-resolution demonstrated that G protein activation efficiency of cone visual pigments is lower than that of rhodopsin, which is one of the molecular bases for the lower amplification of cones compared to rods. In this review, the uniqueness of cone visual pigments is shown by comparison of their molecular properties with those of non-visual opsins and rhodopsin. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

β-arrestin functionally regulates the non-bleaching pigment parapinopsin in lamprey pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a "beta-like" arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

Beta-Arrestin Functionally Regulates the Non-Bleaching Pigment Parapinopsin in Lamprey Pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a “beta-like” arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.     

Activation of Transducin by Bistable Pigment Parapinopsin in the Pineal Organ of Lower Vertebrates

Pineal organs of lower vertebrates contain several kinds of photosensitive molecules, opsins that are suggested to be involved in different light-regulated physiological functions. We previously reported that parapinopsin is an ultraviolet (UV)-sensitive opsin that underlies hyperpolarization of the pineal photoreceptor cells of lower vertebrates to achieve pineal wavelength discrimination. Although, parapinopsin is phylogenetically close to vertebrate visual opsins, it exhibits a property similar to invertebrate visual opsins and melanopsin: the photoproduct of parapinopsin is stable and reverts to the original dark states, demonstrating the nature of bistable pigments. Therefore, it is of evolutionary interest to identify a phototransduction cascade driven by parapinopsin and to compare it with that in vertebrate visual cells. Here, we showed that parapinopsin is coupled to vertebrate visual G protein transducin in the pufferfish, zebrafish, and lamprey pineal organs. Biochemical analyses demonstrated that parapinopsins activated transducin in vitro in a light-dependent manner, similar to vertebrate visual opsins. Interestingly, transducin activation by parapinopsin was provoked and terminated by UV- and subsequent orange-lights irradiations, respectively, due to the bistable nature of parapinopsin, which could contribute to a wavelength-dependent control of a second messenger level in the cell as a unique optogenetic tool. Immunohistochemical examination revealed that parapinopsin was colocalized with Gt2 in the teleost, which possesses rod and cone types of transducin, Gt1, and Gt2. On the other hand, in the lamprey, which does not possess the Gt2 gene, in situ hybridization suggested that parapinopsin-expressing photoreceptor cells contained Gt1 type transducin GtS, indicating that lamprey parapinopsin may use GtS in place of Gt2. Because it is widely accepted that vertebrate visual opsins having a bleaching nature have evolved from non-bleaching opsins similar to parapinopsin, these results implied that ancestral bistable opsins might acquire coupling to the transducin-mediated cascade and achieve light-dependent hyperpolarizing response of the photoreceptor cells.     

Photochemical nature of parietopsin

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Chimeric nature of pinopsin between rod and cone visual pigments.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extra-retinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and approximately 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin II is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.     

Characterization of the primary photointermediates of Drosophila rhodopsin

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.     

Diversity of Active States in TMT Opsins

Opn3/TMT opsins belong to one of the opsin groups with vertebrate visual and non-visual opsins, and are widely distributed in eyes, brains and other internal organs in various vertebrates and invertebrates. Vertebrate Opn3/TMT opsins are further classified into four groups on the basis of their amino acid identities. However, there is limited information about molecular properties of these groups, due to the difficulty in preparing the recombinant proteins. Here, we successfully expressed recombinant proteins of TMT1 and TMT2 opsins of medaka fish (Oryzias latipes) in cultured cells and characterized their molecular properties. Spectroscopic and biochemical studies demonstrated that TMT1 and TMT2 opsins functioned as blue light-sensitive Gi/Go-coupled receptors, but exhibited spectral properties and photo-convertibility of the active state different from each other. TMT1 opsin forms a visible light-absorbing active state containing all-trans-retinal, which can be photo-converted to 7-cis- and 9-cis-retinal states in addition to the original 11-cis-retinal state. In contrast, the active state of TMT2 opsin is a UV light-absorbing state having all-trans-retinal and does not photo-convert to any other state, including the original 11-cis-retinal state. Thus, TMT opsins are diversified so as to form a different type of active state, which may be responsible for their different functions.     

Heterologous expression of limulus rhodopsin

Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments.     

Multiple visual pigments in a photoreceptor of the salamander retina

Although a given retina typically contains several visual pigments, each formed from a retinal chromophore bound to a specific opsin protein, single photoreceptor cells have been thought to express only one type of opsin. This design maximizes a cell''s sensitivity to a particular wavelength band and facilitates wavelength discrimination in retinas that process color. We report electrophysiological evidence that the ultraviolet-sensitive cone of salamander violates this rule. This cell contains three different functional opsins. The three opsins could combine with the two different chromophores present in salamander retina to form six visual pigments. Whereas rods and other cones of salamander use both chromophores, they appear to express only one type of opsin per cell. In visual pigment absorption spectra, the bandwidth at half-maximal sensitivity increases as the pigment''s wavelength maximum decreases. However, the bandwidth of the UV-absorbing pigment deviates from this trend; it is narrow like that of a red-absorbing pigment. In addition, the UV-absorbing pigment has a high apparent photosensitivity when compared with that of red- and blue-absorbing pigments and rhodopsin. These properties suggest that the mechanisms responsible for spectrally tuning visual pigments separate two absorption bands as the wavelength of maximal sensitivity shifts from UV to long wavelengths.     

Sequence, genomic structure and tissue expression of carp (Cyprinus carpio L.) vertebrate ancient (VA) opsin

We report the isolation and characterisation of a novel opsin cDNA from the retina and pineal of the common carp (Cyprinus carpio L.). When a comparison of the amino acid sequences of salmon vertebrate ancient opsin (sVA) and the novel carp opsin are made, and the carboxyl terminus is omitted, the level of identity between these two opsins is 81% and represents the second example of the VA opsin family. We have therefore termed this C. carpio opsin as carp VA opsin (cVA opsin). We show that members of the VA opsin family may exist in two variants or isoforms based upon the length of the carboxyl terminus and propose that the mechanism of production of the short VA opsin isoform is alternative splicing of intron 4 of the VA opsin gene. The VA opsin gene consists of five exons, with intron 2 significantly shifted in a 3' direction relative to the corresponding intron in rod and cone opsins. The position (or lack) of intron 2 appears to be a diagnostic feature which separates the image forming rod and cone opsin families from the more recently discovered non-visual opsin families (pin-opsins (P), vertebrate ancient (VA), parapinopsin (PP)). Finally, we suggest that lamprey P opsin should be reassigned to the VA opsin family based upon its level of amino acid identity, genomic structure with respect to the position of intron 2 and nucleotide phylogeny.     

Spectral tuning by opsin coexpression in retinal regions that view different parts of the visual field

Vision frequently mediates critical behaviours, and photoreceptors must respond to the light available to accomplish these tasks. Most photoreceptors are thought to contain a single visual pigment, an opsin protein bound to a chromophore, which together determine spectral sensitivity. Mechanisms of spectral tuning include altering the opsin, changing the chromophore and incorporating pre-receptor filtering. A few exceptions to the use of a single visual pigment have been documented in which a single mature photoreceptor coexpresses opsins that form spectrally distinct visual pigments, and in these exceptions the functional significance of coexpression is unclear. Here we document for the first time photoreceptors coexpressing spectrally distinct opsin genes in a manner that tunes sensitivity to the light environment. Photoreceptors of the cichlid fish, Metriaclima zebra, mix different pairs of opsins in retinal regions that view distinct backgrounds. The mixing of visual pigments increases absorbance of the corresponding background, potentially aiding the detection of dark objects. Thus, opsin coexpression may be a novel mechanism of spectral tuning that could be useful for detecting prey, predators and mates. However, our calculations show that coexpression of some opsins can hinder colour discrimination, creating a trade-off between visual functions.     

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10–17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.Organisms use color vision for survival behaviors such as foraging, mating, and predator avoidance (1–3). Color vision in invertebrates ranges from trichromatic systems capable of detecting UV, blue, and green (e.g. bees and flies) to the highly complex mantis shrimps (stomatopods) having 12 spectrally distinct classes of photoreceptor cells (4). Despite the diversity of invertebrate color vision systems and the large collection of naturally occurring visual pigments, important questions remain concerning the molecular mechanisms that regulate color sensitivity.In both vertebrates and invertebrates, the visual pigment rhodopsin consists of a chromophore (e.g. 11-cis retinal) covalently bound to an opsin apoprotein via a protonated Schiff base. Upon light absorption, the chromophore isomerizes from cis to all-trans, inducing conformational changes in the opsin that produce activated metarhodopsin. Specific interactions between the retinal chromophore and residues in the opsin tune the λ_max of the chromophore. Studies have shown that Glu-113 (bovine position) serves as the retinylidene Schiff base counter-ion in vertebrate visual pigments (5–7). Removing the negative charge of the counter-ion from the binding pocket deprotonates the chromophore and yields a UV-absorbing pigment (5–7). Using sequence alignments, phylogenetic analysis, analysis of the bovine rhodopsin crystal structure (PDB² entry 1U19), and functional experiments, a large number of amino acids involved in the spectral tuning of vertebrate visual pigments have been identified (8).In contrast, the counter-ion for invertebrate rhodopsin remains unknown, and only one spectrally relevant residue has been identified: an amino acid substitution in Drosophila pigments responsible for UV versus visible sensitivity (9). Interestingly, this amino acid substitution (Gly-90 in bovine rhodopsin) coincides with a substitution that mediates UV versus blue sensitivity in several bird species (10, 11). This discovery highlights the value of a cross-phyla comparison of visual pigments as a means to identify structural differences that may regulate color vision in invertebrates.In the present study, we identify an amino acid substitution in Drosophila visual pigments that regulates the color sensitivity of blue- and green-absorbing rhodopsins. For these studies, we employed sequence analysis of invertebrate and vertebrate visual pigments and a functional examination of mutant invertebrate opsins. This amino acid substitution red-shifts the λ_max of the Drosophila Rh1 pigment and reciprocally blue-shifts the λ_max of Rh6 pigment. Interestingly, this site also affects the spectral tuning of vertebrate pigments and corresponds to Ala-292 in bovine rhodopsin (8, 12–16).     

The opsins

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the G_q-coupled opsin/melanopsin subfamily, the G_o-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.     

Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations

Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.     

Molecular characterization of crustacean visual pigments and the evolution of pancrustacean opsins

Investigations of opsin evolution outside of vertebrate systems have long been focused on insect visual pigments, whereas other groups have received little attention. Furthermore, few studies have explicitly investigated the selective influences across all the currently characterized arthropod opsins. In this study, we contribute to the knowledge of crustacean opsins by sequencing 1 opsin gene each from 6 previously uncharacterized crustacean species (Euphausia superba, Homarus gammarus, Archaeomysis grebnitzkii, Holmesimysis costata, Mysis diluviana, and Neomysis americana). Visual pigment spectral absorbances were measured using microspectrophotometry for species not previously characterized (A. grebnitzkii=496 nm, H. costata=512 nm, M. diluviana=501 nm, and N. americana=520 nm). These novel crustacean opsin sequences were included in a phylogenetic analysis with previously characterized arthropod opsin sequences to determine the evolutionary placement relative to the well-established insect spectral clades (long-/middle-/short-wavelength sensitive). Phylogenetic analyses indicate these novel crustacean opsins form a monophyletic clade with previously characterized crayfish opsin sequences and form a sister group to insect middle-/long-wavelength-sensitive opsins. The reconstructed opsin phylogeny and the corresponding spectral data for each sequence were used to investigate selective influences within arthropod, and mainly "pancrustacean," opsin evolution using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a historical (i.e., phylogenetic) context. Although the conservative dN/dS methods did not detect any selection, 4 amino acid properties (coil tendencies, compressibility, power to be at the middle of an alpha-helix, and refractive index) were found to be influenced by destabilizing positive selection. Ten amino acid sites relating to these properties were found to face the binding pocket, within 4 A of the chromophore and thus have the potential to affect spectral tuning.     

Spectral tuning in salamander visual pigments studied with dihydroretinal chromophores.

In visual pigments, opsin proteins regulate the spectral absorption of a retinal chromophore by mechanisms that change the energy level of the excited electronic state relative to the ground state. We have studied these mechanisms by using photocurrent recording to measure the spectral sensitivities of individual red rods and red (long-wavelength-sensitive) and blue (short-wavelength-sensitive) cones of salamander before and after replacing the native 3-dehydro 11-cis retinal chromophore with retinal analogs: 11-cis retinal, 3-dehydro 9-cis retinal, 9-cis retinal, and 5,6-dihydro 9-cis retinal. The protonated Schiff's bases of analogs with unsaturated bonds in the ring had broader spectra than the same chromophores bound to opsins. Saturation of the bonds in the ring reduced the spectral bandwidths of the protonated Schiff's bases and the opsin-bound chromophores and made them similar to each other. This indicates that torsion of the ring produces spectral broadening and that torsion is limited by opsin. Saturating the 5,6 double bond in retinal reduced the perturbation of the chromophore by opsin in red and in blue cones but not in red rods. Thus an interaction between opsin and the chromophoric ring shifts the spectral maxima of the red and blue cone pigments, but not that of the red rod pigment.     

<Emphasis Type="Italic">In silico</Emphasis> characterisation and chromosomal localisation of human <Emphasis Type="Italic">RRH</Emphasis> (peropsin) – implications for opsin evolution

Background  

The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue,RRH) is unknown.