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PubMed2004

On the relation between the photoactivation energy and the absorbance spectrum of visual pigments.

We relate the collected experimental data on the minimum energy for photoactivation (E(a)) to the wavelengths of peak absorbance (lambda(max)) of 12 visual pigments. The E(a) values have been determined from the temperature-dependence of spectral sensitivity in the long-wavelength range. As shown previously, the simple physical idea E(a) =const. x (1/lambda(max)) (here termed the Stiles-Lewis-Barlow or SLB relation) does not hold strictly. Yet there is a significant correlation between E(a) and 1/lambda(max) (r(2)=0.73) and the regression slope obtained by an unbiased fit is 84% of the predicted value of the best SLB fit. The correlation can be decomposed into effects of A1 --> A2 chromophore change and effects of opsin differences. For a chromophore change in the same opsin, studied in two A1/A2 pigment pairs, the SLB relation holds nearly perfectly. In seven pigments having different opsins but the same (A2) chromophore, the correlation of E(a) and 1/lambda(max) remained highly significant (r(2)=0.78), but the regression coefficient is only 72% of the best SLB fit. We conclude that (1) when the chromophore is exchanged in the same opsin, the lambda(max) shift directly reflects the difference in photoactivation energies, (2) when the opsin is modified by amino acid substitutions, lambda(max) and E(a) can be tuned partly independently, although there is a dominant tendency for inverse proportionality. In four (A1) rhodopsins with virtually the same lambda(max), E(a) varied over a 4.5 kcal/mol range, which may be taken as a measure of the freedom for independent tuning. Assuming that low E(a) correlates with high thermal noise, we suggest that the leeway in lambda(max) - E(a) coupling is used by natural selection to keep E(a) as high as possible in long-wavelength-sensitive pigments, and that this is why the opsin-dependent E(a) (1/lambda(max))-relation is shallower than predicted.

Animals↗
PubMed1994

Expression of octopus rhodopsin in Escherichia coli.

Apoproteins, having various molecular weights, of octopus rhodopsin (oRh, 455 amino acids), which is a typical transmembrane protein, were expressed in Escherichia coli with an inducible expression system using promoter phi 10 of the T7 phage. Fifteen synthetic genes (212-1,365 bp) for oRh (1) were cloned downstream from gene 10 of the T7 phage (846 bp), under the control of promoter phi 10. An expression vector for mature oRh containing no extra peptide resulting from gene 10 was also constructed. Protein productivities were mainly evaluated by ELISA using monoclonal antibodies. The expression level in E. coli of the fused oRh genes varied between 1 and 200 mg/liter of the culture medium (from approximately 0.25 to 50% of the total cell protein, respectively), depending on the fused oRh genes. The amount of mature oRh protein expressed in E. coli was approximately 0.1 to 1 mg/liter. Hydropathy index analysis of gene products showed a significant negative correlation (rho = - 0.63) between expression level of oRh gene products in E. coli and their hydrophobic characteristics. Wavelength shifting of the absorption maximum by exogenous addition of retinal to apoprotein similar to that of authentic oRh was demonstrated in the membrane fraction of E. coli expressing mature opsin.

Animals↗
PubMed1987

Opsin accumulation in photoreceptor inner segment plasma membranes of dystrophic RCS rats.

Opsin localization in photoreceptor plasma membrane was studied in 10- to 30-day-old dystrophic RCS rats. Preembedding cytochemical procedures with antiopsin antibodies and electron microscopy were employed. In the second postnatal week, opsin was sequestered to the outer segment plasma membrane in affected rat retinas. This distribution resembled that observed in the photoreceptors of normal rats at this age. As unphagocytosed debris accumulated in the subretinal space, the outer segments degenerated, and the distribution of opsin in the plasma membrane changed. Opsin reappeared in the inner segment plasma membrane as the outer segments were lost. Loss of the regionalized distribution of opsin was not associated with visible ultrastructural changes in the inner segments or connecting cilium. Severely damaged cells invariably were labeled on their inner segments at high density. Thus, the presence of an outer segment was correlated with the clearance of opsin from the inner segment, while damage to the outer segment was followed by reappearance of opsin in the inner segment plasma membrane.

Animals↗
PubMedDate not supplied

Salamander UV cone pigment: sequence, expression, and spectral properties.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

Amino Acid Sequence↗
PubMed1995

Bovine opsin gene expression exhibits a late fetal to adult regulatory switch.

Rates of bovine photoreceptor gene transcription, as measured by nuclear run-on assays, exhibit gene-specific patterns of regulation. Here we investigate initiation and elongation in nuclear run-on assays with the use of sarkosyl to further understand the nature of these gene-specific elements. Opsin transcription, alone among several genes tested, proved sarkosyl-sensitive. This sensitivity is maximal in adult retinas, with inhibition first detected in mid-third trimester fetal retinas. Therefore, opsin transcription appears to involve different regulatory elements in adult and fetal retinas, implying a fetal to adult switch in the control of opsin gene expression. Although this regulatory switch is initially activated at a time when the fetal outer nuclear layer of the retina first achieves adult-like morphology, further maturation of opsin regulation takes place postpartum since levels of sarkosyl sensitivity are almost 5-fold greater in adult retinas compared to the 7.5 month fetus. We also show that the sarkosyl-induced reduction of opsin transcription is not due to prevention of de novo RNA polymerase II initiation in the run-on reaction, suggesting the detergent alters a positive-acting, postinitiation component of the transcriptional apparatus. Since levels of opsin transcription with sarkosyl are similar to those of the other visual transduction genes with or without sarkosyl, this detergent-sensitive transcriptional component appears to account for the singularly high, gene-specific rate of opsin transcription in retinal photoreceptor cells.

Aging↗
PubMed1995

Opsin phylogeny and evolution: a model for blue shifts in wavelength regulation.

The vast diversity in spectral sensitivities in the vision of many organisms is mediated mostly (although not entirely) through variation in the photosensitive visual pigments (opsins) of the eye. Specifically, shifts in absorption maxima of visual pigments are thought to be a result of interactions within the binding pocket of the opsin, between amino acid side chains and the retinal chromophore, However, it has proven difficult to identify specific amino acid residues important in determining wavelength absorption maxima, especially for some of the short wavelength (blue) opsins. In this paper, a comparative phylogenetic approach was applied to opsin protein sequence data to identify residues important in opsin wavelength regulation. In essence, this approach consisted of interpreting evolutionary history as a series of experiments in which natural selection has repeatedly favored amino acid replacements of certain residues to shift the opsin absorption spectra to either shorter or longer wavelengths. Opsin protein sequences were obtained from GenBank, aligned, and used to reconstruct a phylogenetic tree. Amino acid replacements were traced along the branches of this opsin tree, focusing only on residues likely to reside within the chromophore-binding pocket. A number of functionally convergent, nonconservative amino acid replacements in independently evolved opsins with similar shifts in spectral properties were identified. In short, reconstruction of the phylogeny of the opsin molecule allowed us to track amino acid substitutions in specific sites within the opsin and to target those particular substitutions that are repeatedly associated with marked changes in peak absorbance, shifting the spectral sensitivity of the opsin toward shorter or longer wavelengths. Based on these results, we propose a model for blue shifts of opsin absorption spectra. Amino acid replacements of four polar and charged residues near the protonated Schiff base (SBH+) end of the chromophore are proposed to result in blue shifts of the opsin absorption spectra. This model may explain some of the diversity of blue opsins apparent in both vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed1999

Opsin shift in an aldolase antibody.

An antibody-retinal assembly that mimics the opsin shift (OS) of the naturally occurring visual pigments is reported. Both experiments and calculations show that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which exhibits a remarkable opsin red shift (140 nm), represents a useful model system for studying the factors that contribute to the OS.

Antibodies↗
PubMedDate not supplied

Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse.

The goal of this study was to determine whether the jellyfish green fluorescent protein (GFP) could be used in transgenic mice to label and purify cone photoreceptors from the living retina. We created a transgene containing the 5' regulatory sequence of the human red pigment gene (pR6.5 lacZ clone; kindly provided by J. Nathans & Y. Wang), fused to the GFP coding sequence. This transgene was used to generate seven lines of PCR-positive founders. Three of the lines had bright green fluorescent cone photoreceptors. The GFP fills the entire cell. Two mouse lines had only a few (-10-100) fluorescent cells per retina, and one line (R6.85933) had many thousands. In the latter, double labeling of the cones with RITC-conjugated peanut agglutinin reveals that in the ventral retina a small proportion of the cones express GFP, while in the dorsal retina the majority do. Cells dissociated from the retinae of line R6.85933 continue to fluoresce and can be readily detected and enriched with flow cytometry. The signal provides a log unit of separation between the fluorescent cone soma and the remaining retinal cells. Roughly 3% of the cells are this fluorescent, and it is possible to purify up to 30,000 cells from one mouse. RT-PCR analysis of the mRNA from these isolated cells detects both the middle and short wavelength opsins with little if any contamination from rhodopsin.

Animals↗
PubMed1997

Characterisation of the ultraviolet-sensitive opsin gene in the honey bee, Apis mellifera.

The cDNA sequence of the ultraviolet-sensitive opsin in the honey-bee, Apis mellifera, with associated 5' and 3' untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet-sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven alpha-helical transmembrane regions of insect ultraviolet/blue-sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non-polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet-sensitive pigments represent an ancient class of insect opsins.

Amino Acid Sequence↗
PubMed2005

Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development.

Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4) in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.

Animals↗
PubMed2006

The spectral sensitivity of the lens eyes of a box jellyfish, Tripedalia cystophora (Conant).

Box jellyfish, or cubomedusae (class Cubozoa), are unique among the Cnidaria in possessing lens eyes similar in morphology to those of vertebrates and cephalopods. Although these eyes were described over 100 years ago, there has been no work done on their electrophysiological responses to light. We used an electroretinogram (ERG) technique to measure spectral sensitivity of the lens eyes of the Caribbean species Tripedalia cystophora. The cubomedusae have two kinds of lens eyes, the lower and upper lens eyes. We found that both lens eye types have similar spectral sensitivities, which likely result from the presence of a single receptor type containing a single opsin. The peak sensitivity is to blue-green light. Visual pigment template fits indicate a vitamin A-1 based opsin with peak sensitivity near 500 nm for both eye types.

Animals↗
PubMed1988

Guanosine triphosphate promotes the post-translational integration of opsin into the endoplasmic reticulum membrane.

Membrane integration of a nascent opsin polypeptide was examined to determine whether insertion of proteins into the endoplasmic reticulum is dependent upon energy provided by ribonucleotide triphosphate hydrolysis. A discrete-sized nascent chain was obtained by in vitro translation of a mRNA which lacked a termination codon yet encoded the first 156 residues of bovine opsin. Ribosomes bearing the newly synthesized opsin chains were post-translationally incubated with canine pancreas microsomal membrane vesicles after addition of exogenous ribonucleotides or ribonucleotide analogues. Post-translational membrane integration and glycosylation of the 156-residue nascent polypeptide was found to require either the presence of guanosine triphosphate or a nonhydrolyzable GTP analogue. ATP did not promote post-translational integration of the nascent polypeptide. Although ribonucleotide hydrolysis was not obligatorily required for integration of opsin, we observed an increase in the proportion of glycosylated opsin chains in post-translational incubations that contained hydrolyzable ribonucleotide triphosphates. We conclude that a GTP-binding protein performs an essential role during integration of opsin into the endoplasmic reticulum.

Animals↗
PubMed1988

Acidic fibroblast growth factor stimulates opsin levels in retinal photoreceptor cells in vitro.

It is demonstrated that newborn rat retinal photoreceptor cells can differentiate in monolayer culture, and synthesize de novo photoreceptor-specific proteins such as opsin. When maintained in serum supplemented medium on a laminin substrate, these cells survive for up to 3 weeks. The addition of acidic fibroblast growth factor stimulates an increase in the levels of opsin of 5-10-fold control values, and prolongs cell survival by up to 6 days.

Animals↗
PubMed1996

Phylogenetic relationships among short wavelength-sensitive opsins of American chameleon (Anolis carolinensis) and other vertebrates.

The vertebrate opsins have been classified into four major phylogenetic groups. One of them, a short wavelength-sensitive (SWS) opsin group, is further divided into two subgroups, SWS-I and SWS-II, having the wavelengths of maximal absorption of about 420 and 450 nm, respectively. Here we report the DNA sequences of the SWS-I and SWS-II genes from the lizard Anolis carolinensis. The shorter wavelengths of absorption by the two SWS subgroup opsins seem to be achieved by different sets of amino acid replacements in the transmembrane regions.

Amino Acid Sequence↗
PubMed2006

Exploring the molecular mechanism for color distinction in humans.

We examine here the role of the red, green, and blue human opsin structures in modulating the absorption properties of 11-cis-retinal bonded to the protein via a protonated Schiff base (PSB). We built the three-dimensional structures of the human red, green, and blue opsins using homology modeling techniques with the crystal structure of bovine rhodopsin as the template. We then used quantum mechanics (QM) combined with molecular mechanics (MM) (denoted as QM/MM) techniques in conjunction with molecular dynamics to determine how the room temperature molecular structures of the three human color opsin proteins modulate the absorption frequency of the same bound 11-cis-retinal chromophore to account for the differences in the observed absorption spectra. We find that the conformational twisting of the 11-cis-retinal PSB plays an important role in the green to blue opsin shift, whereas the dipolar side chains in the binding pocket play a surprising role of red-shifting the blue opsin with respect to the green opsin, as a fine adjustment to the opsin shift. The dipolar side chains play a large role in the opsin shift from red to green.

Amino Acid Sequence↗
PubMed2001

Functional diversification of lepidopteran opsins following gene duplication.

A comparative approach was taken for identifying amino acid substitutions that may be under positive Darwinian selection and are correlated with spectral shifts among orthologous and paralogous lepidopteran long wavelength-sensitive (LW) opsins. Four novel LW opsin fragments were isolated, cloned, and sequenced from eye-specific cDNAs from two butterflies, Vanessa cardui (Nymphalidae) and Precis coenia (Nymphalidae), and two moths, Spodoptera exigua (Noctuidae) and Galleria mellonella (Pyralidae). These opsins were sampled because they encode visual pigments having a naturally occurring range of lambda(max) values (510-530 nm), which in combination with previously characterized lepidopteran opsins, provide a complete range of known spectral sensitivities (510-575 nm) among lepidopteran LW opsins. Two recent opsin gene duplication events were found within the papilionid but not within the nymphalid butterfly families through neighbor-joining, maximum parsimony, and maximum likelihood phylogenetic analyses of 13 lepidopteran opsin sequences. An elevated rate of evolution was detected in the red-shifted Papilio Rh3 branch following gene duplication, because of an increase in the amino acid substitution rate in the transmembrane domain of the protein, a region that forms the chromophore-binding pocket of the visual pigment. A maximum likelihood approach was used to estimate omega, the ratio of nonsynonymous to synonymous substitutions per site. Branch-specific tests of selection (free-ratio) identified one branch with omega = 2.1044, but the small number of substitutions involved was not significantly different from the expected number of changes under the neutral expectation of omega = 1. Ancestral sequences were reconstructed with a high degree of certainty from these data. Reconstructed ancestral sequences revealed several instances of convergence to the same amino acid between butterfly and vertebrate cone pigments, and between independent branches of the butterfly opsin tree that are correlated with spectral shifts.

Amino Acid Sequence↗