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PubMed1999

Photons, femtoseconds and dipolar interactions: a molecular picture of the primary events in vision.

The first 200 femtoseconds in the life of a photoexcited rhodopsin molecule are extremely important for the development of visual sensation. Immediately upon excitation, a dramatic change in the charge distribution of the cationic 11-cis-retinal protonated Schiff base chromophore occurs that is quantitated by the change in electronic dipole moment of approximately 15 Debye. The opsin protein tunes the absorption maximum of the pigment to the blue or to the red enabling colour vision by placing dipolar rather than charged residues in the chromophore binding site to differentially stabilize either the ground or the excited state charge distribution. Resonance Raman intensity analysis reveals that the 11-cis-retinal chromophore then distorts violently about the C11 = C12 double bond, reaching torsional angles of approximately 70 degrees in only 30 fs. This rapid torsional distortion is driven by the non-bonded interaction between the 13-methyl group and the 10-hydrogen that is unique to the 11-cis configuration of the chromophore. The excited state depopulates in approximately 50 fs through a rapid and vibrationally coherent transition to the ground electronic state manifold with relaxation to the formally trans photoproduct complete in only 200 fs. This unusually fast and efficient isomerization process establishes a new paradigm for condensed phase photochemical reactions.

Animals↗
PubMed2003

Phylogeny of bumble bees in the New World subgenus Fervidobombus (Hymenoptera: Apidae): congruence of molecular and morphological data.

We present new DNA sequence data (12S, 16S, and opsin gene fragments) and morphological characters of the male genitalia for a phylogenetic analysis of the bumble bee subgenus Fervidobombus. There is no significant incongruence between the three molecular data sets, and little incongruence between the DNA and morphology. Simultaneous analysis of all the data partitions resulted in a tree that was entirely congruent with the All-DNA tree. Optimization of the geographic locations of the taxa onto the tree topology using dispersal/vicariance analysis suggests a complex picture of spread and diversification of Fervidobombus from the Old World into the southern New World. There is a phylogenetic component to their spread into tropical rain forest, as the two primary rain forest species (Bombus transversalis and Bombus pullatus) comprise a monophyletic clade, along with a third species, Bombus atratus, which is widely distributed in South America, including lowland subtropical habitats.

Animals↗
PubMed2001

On the bioactive conformation of the rhodopsin chromophore: absolute sense of twist around the 6-s-cis bond.

Incubation of opsin with synthetic 6-s-locked retinoids 2a and 2b only led to pigment formation from the alpha-locked 2a, the CD spectrum of which was similar to that of native rhodopsin (Rh). This establishes that the 6-s-bond of the chromophore in rhodopsin is cis, and that its helicity is negative. Earlier cross-linking studies showed that the 11-cis to all-trans photoisomerization occurring in the batho-Rh to lumi-Rh conversion induces a flip over of the side carrying the ring moiety. The GTP-binding assay of pigment Rh-(2a), incorporating retinal analogue 2a, has shown that its activity is 80% that of the native pigment. That is, the overall conformation around the 6-s bond is retained in the steps leading to G-protein activation.

Animals↗
PubMed2004

Extensive duplications of phototransduction genes in early vertebrate evolution correlate with block (chromosome) duplications.

Many gene families in mammals have members that are expressed more or less uniquely in the retina or differentially in specific retinal cell types. We describe here analyses of nine such gene families with regard to phylogenetic relationships and chromosomal location. The families are opsins, G proteins (alpha, beta, and gamma subunits), phosphodiesterases type 6, cyclic nucleotide-gated channels, G-protein-coupled receptor kinases, arrestins, and recoverins. The results suggest that multiple new gene copies arose in all of these families very early in vertebrate evolution during a period with extensive gene duplications. Many of the new genes arose through duplications of large chromosome regions (blocks of genes) or even entire chromosomes, as shown by linkage with other gene families. Some of the phototransduction families belong to the same duplicated regions and were thus duplicated simultaneously. We conclude that gene duplications in early vertebrate evolution probably helped facilitate the specialization of the retina and the subspecialization of different retinal cell types.

Animals↗
PubMed2004

Opsin photoisomerases in the chick retina and pineal gland: characterization, localization, and circadian regulation.

PURPOSE: The chick retina and pineal gland exhibit circadian oscillations in biochemical and physiological processes in vivo and in vitro, which entrain to 24-hour light-dark cycles. However, the phototransduction mechanisms responsible for entrainment are largely unknown. The present study characterizes two candidate opsinlike genes that may play a role in entrainment of the clocks in these tissues. METHODS: Bioinformatics, cladistic techniques, and in situ hybridization and Northern blot analyses were conducted to characterize, localize, and determine the circadian expression of the candidate opsinlike genes in the retina and pineal gland. RESULTS: Two candidate photosensors and/or photoisomerases were predominantly distributed within the pineal gland and retina: the retinal pigmented epithelium-derived rhodopsin homologue (peropsin, gRrh) and retinal G-protein-coupled receptor opsin (RGR opsin, gRgr). Northern blot and in situ analyses revealed mRNA expression for both opsins in the pineal gland, retina, and brain tissue. The mRNA for both genes within the pineal gland and retina is regulated on a circadian basis such that they are highest in late subjective day. Digoxigenin in situ analyses showed retinal gRgr message within the inner nuclear layer (INL) and retinal ganglion cell layer (RGL), whereas gRrh message was distributed predominantly in the RGL. In the pineal gland, gRgr message was sparsely distributed among pinealocytes in follicles, but not within the follicles themselves, whereas gRrh was localized in interstitial areas indicative of astrocytic and/or endothelial origin. CONCLUSIONS: The presence of two putative photoisomerases within the pineal gland and in retinal layers associated with biological clock function provides two candidate opsinlike genes that may serve in the visual cycle regulation of the circadian clock.

Amino Acid Sequence↗
PubMed1995

Galactosylation of rhodopsin by the human retina.

The major oligosaccharide chains of bovine, frog and human rhodopsins are abridged, hybrid, asparagine-linked structures that contain only mannose and N-acetylglucosamine as the constituent sugars. Isomers that also contain galactose have been detected in varying amounts in rhodopsins from different species. As part of studies to examine the mechanisms used by the human retina to control the glycosylation of rhodopsin, the kinetics of the retinal galactosyltransferase was examined using Golgi-enriched fractions of human retina as the enzyme source and rhodopsin, opsin, and the oligosaccharide isolated from rhodopsin as acceptors. These reactions were compared to those using bovine retina and rat liver Golgi. A comparison of the Vmax/Km ratios revealed that the efficiency of the human retinal galactosyltransferase was some 20-fold lower than that of the rat liver. In addition, consistent with the higher state of galactosylation of human as compared to bovine rhodopsin, greater efficiency was observed with the human preparation. While the conformation of the visual pigment exerted an effect, its low galactosylation was not due to a major directing influence on glycosylation by the polypeptide matrix as indicated by the even lower activity toward the isolated oligosaccharide. The galactosylated oligosaccharides obtained by these procedures were identified by chromatographic methods. The relatively low activity of the retinal galactosyltransferases observed in this study may help explain the limited glycosylation that is typical of rhodopsin.

Animals↗
PubMed1997

The molecular evolution of visual pigments of freshwater crayfishes (Decapoda: Cambaridae).

This study examines the diverse maximum wavelength absorption (lambdamax) found in crayfishes (Decapoda: Cambaridae and Parastacidae) and the associated genetic variation in their opsin locus. We measured the wavelength absorption in the photoreceptors of six species that inhabit environments of different light intensities (i.e., burrows, streams, standing waters, and subterranean waters). Our results indicate that there is relatively little variation in lambdamax (522-530 nm) among species from different genera and families. The existing variation did not correlate with the habitat differences of the crayfishes studied. We simultaneously sequenced the rhodopsin gene to identify the amino acid replacements that affect shifts in maximum wavelength absorption. We then related these to changes that correlated with shifts in lambdamax by reconstructing ancestral character states using a maximum-likelihood approach. Using amino acid sequences obtained from five species (all were 301 amino acids in length), we identified a number of candidates for producing shifts of 4 to 8 nm in lambdamax. These amino acid replacements occurred in similar regions to those involved in spectral shifts in vertebrates.

Amino Acid Sequence↗
PubMed2001

Richer color experience in observers with multiple photopigment opsin genes.

Traditional color vision theory posits that three types of retinal photopigments transduce light into a trivariate neural color code, thereby explaining color-matching behaviors. This principle of trichromacy is in need of reexamination in view of molecular genetics results suggesting that a substantial percentage of women possess more than three classes of retinal photopigments. At issue is the question of whether four-photopigment retinas necessarily yield trichromatic color perception. In the present paper, we review results and theory underlying the accepted photoreceptor-based model of trichromacy. A review of the psychological literature shows that gender-linked differences in color perception warrant further investigation of retinal photopigment classes and color perception relations. We use genetic analyses to examine an important position in the gene sequence, and we empirically assess and compare the color perception of individuals possessing more than three retinal photopigment genes with those possessing fewer retinal photopigment genes. Women with four-photopigment genotypes are found to perceive significantly more chromatic appearances in comparison with either male or female trichromat controls. We provide a rationale for this previously undetected finding and discuss implications for theories of color perception and gender differences in color behavior.

Adult↗
PubMed1985

Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity.

Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.

Animals↗
PubMed1999

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line.

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.

Amino Acid Sequence↗
PubMed2003

Cone deactivation kinetics and GRK1/GRK7 expression in enhanced S cone syndrome caused by mutations in NR2E3.

PURPOSE: To determine the relationship between cone deactivation kinetics in patients with the enhanced S cone syndrome (ESCS) caused by mutations in NR2E3 and the immunoreactivity to G-protein-coupled receptor kinase 1 (GRK1) and GRK7. METHODS: Electroretinogram (ERG) photoresponses were used to investigate activation kinetics of cones with a model of cone phototransduction. Deactivation kinetics of cones after bright flashes was quantified with a paired-flash ERG paradigm. Immunocytochemistry was performed with antibodies against cone opsins and kinases GRK1 and GRK7 in postmortem normal and ESCS retinal tissue. RESULTS: Activation kinetics of long/middle-wavelength-sensitive (L/M) cone-mediated responses in patients with ESCS were similar to those of normal L/M cones. Activation kinetics of ESCS short-wavelength-sensitive (S) cones, when compared with normal L/M cone responses evoked by the same stimulus, were slower by an amount consistent with the expected differences in spectral sensitivities. After bright flashes chosen to evoke identical activation kinetics, ESCS S cones deactivated much more slowly than ESCS or normal L/M cones. Normal human retina revealed strongly labeled cone outer segments with anti-GRK1 and anti-GRK7. In an ESCS retina, outer segments positive for L/M opsin were strongly labeled with anti-GRK1, whereas outer segments positive for S opsin showed no detectable GRK1 reactivity. GRK7 labeling was absent in all photoreceptors of the ESCS retina. CONCLUSIONS: The cone-dominant human retina resulting from NR2E3 mutations affords greater understanding of the physiological roles of GRK1 and GRK7 in human cone photoreceptors. Normal deactivation kinetics in human L/M cones can occur without GRK7 when GRK1 is present in ESCS, but does not occur when GRK7 is present but GRK1 is deficient in Oguchi disease. Lack of both GRK1 and GRK7 in S cones of patients with ESCS results in a more pronounced abnormality in deactivation kinetics and suggests the existence of partial compensation by either GRK when the other is deficient.

Electroretinography↗
PubMed1999

The endogenous chromophore of retinal G protein-coupled receptor opsin from the pigment epithelium.

The recent identification of nonvisual opsins has revealed an expanding family of vertebrate opsin genes. The retinal pigment epithelium (RPE) and Müller cells contain a blue and UV light-absorbing opsin, the RPE retinal G protein-coupled receptor (RGR, or RGR opsin). The spectral properties of RGR purified from bovine RPE suggest that RGR is conjugated in vivo to a retinal chromophore through a covalent Schiff base bond. In this study, the isomeric structure of the endogenous chromophore of RGR was identified by the hydroxylamine derivatization method. The retinaloximes derived from RGR in the dark consisted predominantly of the all-trans isomer. Irradiation of RGR with 470-nm monochromatic or near-UV light resulted in stereospecific isomerization of the bound all-trans-retinal to an 11-cis configuration. The stereospecificity of photoisomerization of the all-trans-retinal chromophore of RGR was lost by denaturation of the protein in SDS. Under the in vitro conditions, the photosensitivity of RGR is at least 34% that of bovine rhodopsin. These results provide evidence that RGR is bound in vivo primarily to all-trans-retinal and is capable of operating as a stereospecific photoisomerase that generates 11-cis-retinal in the pigment epithelium.

Animals↗
PubMed2006

Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles.

Non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subsequently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles.

Amino Acid Sequence↗
PubMed1999

Volvoxrhodopsin, a light-regulated sensory photoreceptor of the spheroidal green alga Volvox carteri.

Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls the organism's phototactic behavior via two independent photoreceptor currents. Here, we report the identification of an opsinlike gene, designated as volvoxopsin (vop). The encoded protein exhibits homologies to the opsin of the unicellular alga Chlamydomonas reinhardtii (chlamyopsin) and to the entire animal opsin family, thus providing new perspectives on opsin evolution. Volvoxopsin accumulates within the eyes of somatic cells. However, the vop transcript is detectable only in the reproductive eyeless gonidia and embryos. vop mRNA levels increase 400-fold during embryogenesis, when embryos develop in darkness, whereas the vop transcript does not accumulate when embryos develop in the light. An antisense transformant, T3, was generated. This transformant produces 10 times less volvoxopsin than does the wild type. In T3, the vop transcript is virtually absent, whereas the antisense transcript is predominant and light regulated. It follows that vop expression is under light-dependent transcriptional control but that volvoxopsin itself is not the regulatory photoreceptor. Transformant T3 is phototactic, but its phototactic sensitivity is reduced 10-fold relative to the parental wild-type strain HK10. Thus, we offer definitive genetic evidence that a rhodopsin serves as the photoreceptor for phototaxis in a green alga.

Algal Proteins↗
PubMed2005

S-cones do not contribute to the OFF-midget pathway in the retina of the marmoset, Callithrix jacchus.

It is well established that in primate retina both medium- and long-wavelength-sensitive cone types provide input to the midget-parvocellular pathway. The question, however, whether short-wavelength-sensitive (S or 'blue') cones provide input to the OFF-division of the midget-parvocellular pathway is still controversial. In the present study, we investigated the connections of nearly 400 S-cones with OFF-midget bipolar cells in central and peripheral retina of a New World monkey, the marmoset. Horizontal sections or pieces of whole retinae were double-labelled with an antiserum to S-cone opsin to identify S-cones and antibodies to the cell adhesion molecule CD15 to identify OFF-midget bipolar cells. Peanut agglutinin coupled to a fluorescent tag was used to label the cone pedicles of all cone types. Peanut agglutinin was also used to distinguish S-cones from the other cone types. The sections were analysed with deconvolution microscopy. We found that nearly all pedicles of medium- and long-wavelength-sensitive cones are located opposite distinct dendritic clusters formed by OFF-midget bipolar cells. By contrast, the S-cone pedicles are not located opposite dendritic clusters. Instead, S-cones make sparse contacts with CD15-labelled processes. Some of these processes protruded from OFF-midget bipolar clusters, whereas others could be traced to a diffuse bipolar cell type. Thus, in the marmoset retina the midget-parvocellular system does not carry a blue-OFF signal.

Animals↗
PubMed1996

Functional interaction of transmembrane helices 3 and 6 in rhodopsin. Replacement of phenylalanine 261 by alanine causes reversion of phenotype of a glycine 121 replacement mutant.

Replacement of a highly conserved glycine residue on transmembrane (TM) helix 3 of bovine rhodopsin (Gly121) by amino acid residues with larger side chains causes a progressive blue-shift in the lambdamax value of the pigment, a decrease in thermal stability, and an increase in reactivity with hydroxylamine. In addition, mutation of Gly121 causes a relative reversal in the selectivity of opsin for 11-cis-retinal over all-trans-retinal. It was suggested that Gly121 plays an important role in defining the 11-cis-retinal binding pocket of rhodopsin (Han, M., Lin, S. W., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32330-32336). Here, we combined the mutant opsin G121L with second site replacements of four different amino acid residues on TM helix 6: Met257, Val258, Phe261, or Trp265. We show that the loss of function phenotypes of the G121L mutant described above can be partially reverted specifically by the mutation of Phe261, a residue highly conserved in all G protein-coupled receptors. For example, the double-replacement mutant G121L/F261A has spectral, chromophore-binding, and transducin-activating properties intermediate between those of G121L and rhodopsin. This rescue of the G121L defects did not occur with the other second site mutations tested. We conclude that specific portions of TM helices 3 and 6, which include Gly121 and Phe261, respectively, define the chromophore-binding pocket in rhodopsin. Finally, the results are placed in the context of a molecular graphics model of the TM domain of rhodopsin, which includes the retinal-binding pocket.

Alanine↗
PubMed2003

New fluorescent probes for visual proteins. Part II. 5-(Oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate.

A new dual-fluorescent compound, 5-(oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate (1), a derivative of dimethylaminobenzoic acid, has been synthesised and studied photophysically. This compound continues the series of potential fluorescent probes for visual and proton-pumping opsin proteins. The photophysical behaviour of this molecule, including charge-transfer interaction in the ground state and dual-fluorescence emission, is similar to that of the previously studied analogue cis-3-(oxo)propenyl-p-(N,N-dimethylamino)benzoate (cis-2). The presence of several theoretically calculated conformers of compound 2 was suggested to be responsible for the observed strongly red-shifted absorption and excitation wavelength dependence. These photophysical anomalies were also observed for molecule 1, though the models put forward to explain them in the cases of 1 and 2 are rather different. Based on theoretical calculations and experimental results, we propose that some of the stable conformers might be connected with either a charge-transfer complex or mesomeric interactions in the ground state. Upon changing the electronic nature of the oxo-pentadienyl acceptor moiety, e.g. protonation, chemical or biochemical reaction, the charge-transfer absorption disappears, which leads to a dramatic increase in the fluorescence quantum yield.

Animals↗
PubMed1999

The molecular genetics of red and green color vision in mammals.

To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red pigment of goldfish (Carassius auratus), which has a lambdamax value at 559 +/- 4 nm. Multiple linear regression analyses show that S180A, H197Y, Y277F, T285A, and A308S shift the lambdamax values of the red and green pigments in mammals toward blue by 7, 28, 7, 15, and 16 nm, respectively, and the reverse amino acid changes toward red by the same extents. The additive effects of these amino acid changes fully explain the red-green color vision in a wide range of mammalian species, goldfish, American chameleon (Anolis carolinensis), and pigeon (Columba livia).

Amino Acid Sequence↗