Absorption Spectrum Of Rhodopsin Research Articles

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer

Membrane proteins often require solubilization to study their structure or define the mechanisms underlying their function. In this study, the functional properties of the membrane protein rhodopsin in its native lipid environment were investigated after being solubilized with styrene-maleic acid (SMA) copolymer. The static absorption spectra of rhodopsin before and after the addition of SMA were recorded at room temperature to quantify the amount of membrane protein solubilized. The samples were then photobleached to analyze the functionality of rhodopsin upon solubilization. Samples with low or high SMA/rhodopsin ratios were compared to find a threshold in which the maximal amount of active rhodopsin was solubilized from membrane suspensions. Interestingly, whereas the highest SMA/rhodopsin ratios yielded the most solubilized rhodopsin, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation. The results confirm that SMA is a useful tool for membrane protein research, but SMA added in excess can interfere with the dynamics of protein activation.

Light damage to the retina: an historical approach.

A brief review of retinal light damage is presented. Thermal damage requires a local rise in temperature of at least 10 °C, causing an instant denaturation of proteins. The primary absorber is melanin. Photochemical damage occurs at body temperature and involves cellular damage by reactive forms of oxygen. The photosensitizers are photoproducts of the visual pigments. First indications that non-thermal damage might exist, in particular in the case of eclipse blindness, was presented by Vos in 1962. Attribution thereof to photochemical action was presented in 1966 by Noell et al who also measured the first action spectrum, in rat. It turned out to be identical to the absorption spectrum of rhodopsin. However, in 1976 and 1982 Ham et al found a quite different spectrum in monkeys, peaking at short wavelengths. The latter spectrum, but not the former, was confirmed since in numerous publications with animal models including rat. In ophthalmological practice a 'sunburn' was at first the only complaint caused by light damage. To avoid this, patients with dilated pupils should always be advised to wear sunglasses. Since the invention of the laser accidents have been reported, the most recent development is youth playfully pointing a strong laser pen in their eyes with marked consequences. The operation microscope and endoilluminators should always be used as brief as possible to avoid photochemical damage. Arguments for implant lenses that block not only the UV but also part of the visible spectrum seem too weak to justify extra costs.

In vivo imaging rhodopsin distribution in the photoreceptors with nano-second pulsed scanning laser ophthalmoscopy.

Rhodopsin is a biomarker for the function of rod photoreceptors, the dysfunction of which is related to many blinding diseases like retinitis pigmentosa and age-related macular degeneration. Imaging rhodopsin quantitatively may provide a powerful clinical tool for diagnosis of these diseases. To map rhodopsin distribution accurately in the retina, absorption by rhodopsin intermediates need to be minimized. We developed nano-second pulsed scanning laser ophthalmoscopy (SLO) to image rhodopsin distribution in the retina. The system takes advantage of the light-induced shift of rhodopsin absorption spectra, which in turn affects the fundus spectral reflection before and after photo-bleaching. By imaging the retina twice, one in the dark-adapted state and the other one in the light-adapted state, the rhodopsin absorption change can be calculated from the differential image, which is a function of the rhodopsin concentration in the rod photoreceptors. The system was successfully applied to in vivo imaging of rat retina in different bleaching conditions to verify its feasibility. Our studies showed that the differential image between the dark- and light-adapted states represents rhodopsin distribution in the retina. We also conducted a dynamic bleaching experiment to prove the importance of reducing light absorption of rhodopsin intermediates. The preliminary results showed that our nano-second pulsed-light SLO is promising in imaging the functional biomarker of the rod photoreceptors. By using nanosecond pulsed laser, in which one laser pulse generates one pixel of the image, the absorption of rhodopsin intermediates can be reduced.

Femtosecond dynamics of primary processes in visual pigment rhodopsin

The dynamics of the coherent photoisomerization of the 11-cis-retinal in bovine rhodopsin is studied by femtosecond time-resolved laser absorption spectroscopy with a resolution of 30 fs. Rhodopsin is excited with 500-, 535-, and 560-nm femtosecond pulses to produce different initial Franck-Condon states with different vibrational energies of the molecule in its electronically excited state. The time evolution of the photoinduced differential absorption spectra of rhodopsin is measured upon excitation with a femtosecond pulse in a spectral range from 400 to 720 nm. Oscillations in the time-resolved absorption of the rhodopsin photoproducts, such as photorhodopsin with a vibrationally excited all-trans-retinal and in its initial-state rhodopsin with a vibrationally excited 11-cis-retinal, are examined. It is demonstrated that these oscillations reflect the dynamics of coherent vibrational wavepackets. The Fourier transform of these oscillatory components yields frequencies, amplitudes, and initial phases of various vibrational modes involved in the motion the wavepackets in both photoproducts. The main vibrational modes manifest themselves at frequencies of 62 and 160 cm−1 for photorhodopsin and 44 and 142 cm−1 for initial-state rhodopsin. It is shown that these vibrational modes are directly involved in the coherent reaction under the study, with their amplitudes in the power spectrum produced by the Fourier transform of the kinetic curves being dependent on the wavelength of rhodopsin excitation.

The Action Spectrum of Photochemical Damage to the Retina: A Review of Monochromatic Threshold Data

Photochemical damage to the retina occurs for prolonged exposures of intense light. Two action spectra exist for this phenomenon. In rat an action spectrum matching the absorption spectrum of rhodopsin was found. In macaque, the susceptibility for photochemical damage decreased continuously from the UV to long visible wavelengths. Later, such a spectrum was also found in rat. In search for critical parameters that determine the shape of the spectrum we gathered all available data on the damage threshold dose for monochromatic radiation and noted the experimental conditions. The rhodopsin action spectrum was found in two sources; the other 16 sources adhered to the short wavelength spectrum. Comparing the conditions we conclude that the critical parameters for the generation of either action spectrum remain elusive. Experiments are suggested to resolve this issue and fill a few gaps in our knowledge.

Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin

The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.

Vertebrate Rhodopsin Adaptation to Dim Light via Rapid Meta-II Intermediate Formation

Rhodopsin is a photoreceptive protein present in vertebrate rod photoreceptor cells, which are responsible for scotopic vision. Recent molecular studies have shown that several aquatic vertebrate species have independently acquired rhodopsin containing Asp83Asn, Glu122Gln, and Ala292Ser substitutions, causing a blue shift in the rhodopsin absorption spectra for adaptation to the blue-green photic environment in deep water. Here, we provide new evidence for the evolutionary and functional relevance of the Asp83Asn substitution. Spectroscopic and kinetic analyses of rhodopsins in six cichlid fishes from the East African Great Lakes using charge-coupled device spectrophotometer revealed that the Asp83Asn substitution accelerated the formation of meta-II, a rhodopsin intermediate crucial for activation of the G-protein transducin. Because rapid formation of meta-II likely results in effective transduction of photic signals, it is reasonable to assume that deep-water cichlid species have acquired rhodopsin containing Asn83 to adapt to dim lighting. Remarkably, rhodopsin containing Asn83 has been identified in terrestrial vertebrates such as bats, and these rhodopsin variants also exhibit accelerated meta-II formation. Our results indicated that the Asp83Asn substitution observed in a variety of animal species was acquired independently in many different lineages during vertebrate evolution for adaptation to dimly lit environments.

Eyeshine and spectral tuning of long wavelength-sensitive rhodopsins: no evidence for red-sensitive photoreceptors among five Nymphalini butterfly species

Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.

Exploring the Opsin shift with ab initio methods: Geometry and counterion effects on the electronic spectrum of retinal

To study the effect of the charged chromophore environment on the absorption spectrum of rhodopsin, we have calculated excited state energies of chromophore models using multi-configurational second-order perturbation theory. Taking advantage of the recently solved crystal structures of rhodopsin we have considered different chromophore geometries and their interaction with the Glu113 counterion, water and Glu181 in different protonation states. We observe a strongly blueshifted S1 state upon inclusion of Glu113/water to the wave function; the effect of the additional carboxyl group appears to be overbalanced by the complex counterion.

Aberrant photon responses in rods of the macaque monkey

Recovery from bright light was studied in macaque rods by measuring the membrane current of single outer segments. The recovery phase of some responses displayed a plateau current of about one picoampere lasting for several seconds. The following evidence suggests these "steps" are single photon responses of abnormally long duration. (1) Over a limited range of intensities, step amplitude remained constant and summed linearly with intensity. The collecting area for step generation was about 2.6 x 10(-3) microns2. (2) Step duration varied exponentially with a mean duration of about 6.5 s. (3) Fluctuation analysis of the tail currents was consistent with the idea that a step is evoked by isomerization of a single rhodopsin molecule, and that only 1 in 400 isomerizations leads to a responses with a step-like waveform. (4) With only the distal portion of the outer segment in the electrode, the polarity of the step response reversed when the proximal portion of the outer segment was illuminated, indicating that step generation results from a local change in outer segment conductance near the site of photon absorption. (5) The probability of eliciting a step varied with the wavelength of light in the manner expected from the absorption spectrum of rhodopsin.

Light-sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole.

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380-1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 microW/cm2, delta lambda = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca(2+)-dependent. K(+)-rich Hanks' solution, ouabain (inhibitor of Na(+)-K(+)-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells.

Light‐sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380–1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 μW/cm2, Δλ = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca2+-dependent. K+-rich Hanks' solution, ouabain (inhibitor of Na+-K+-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells. © 1996 Wiley-Liss, Inc.

Blue through UV polarization sensitivities in insects

A Signal-to-Noise optimization model has now been extended to explain the range of species-specific polarization sensitivities of insects. The different polarization sensitivities are shown to represent optimizations for the detection of plane polarized (Rayleigh-scattered) skylight over a range of atmospheric polarization conditions. Rhodopsin absorption spectra with peaks in the Blue (λmax≈450 nm) maximize detection efficiencies under conditions of high polarization. Rhodopsin absorption spectra peaking in the UV (λmax≈350 nm) maximize Signal-to-Noise Ratios for the detection of polarized skylight at the other extreme of low polarization.

In vitro identification of rhodopsin in the green alga Chlamydomonas

The unicellular alga Chlamydomonas can detect both intensity and direction of the ambient light and adjust its swimming speed and direction accordingly. On the basis of physiological experiments, the functional photoreceptor for this visual process has recently shown to be a rhodopsin. We here report the in vitro identification of endogenous retinal and a rhodopsin in Chlamydomonas cell extracts and purified membrane preparations. The rhodopsin absorption spectrum has fine structure with the maximum at 495 nm and matches the action spectra for the behavioral light responses. The rhodopsin can be bleached and subsequently reconstituted with exogenous retinal. Labeling with [3H]retinal occurs in the final preparation only with a single protein with a molecular weight of 32,000. We conclude that this protein is the visual photoreceptor in Chlamydomonas.

Rhodopsin Mutants That Bind But Fail to Activate Transducin

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.

We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.

Absorption spectrum of rhodopsin in rod outer segment suspension: role of sieve effect

Absorption spectrum of rhodopsin in rod outer segment suspension: role of sieve effect

The Effect of Screening Pigment Migration on Spectral Sensitivity in a Crayfish Reflecting Superposition Eye

ABSTRACT In crayfish, there is some evidence that visual pigments in the eye affect the spectral sensitivity, both at the receptor level (Goldsmith & Fernandez, 1968; Kong & Goldsmith, 1977) and in the optic fibres (Woodcock & Goldsmith, 1970). However, there is a discrepancy between the absorption spectrum of rhodopsin as measured by microspectrophotometry (λmax is 530–550 nm, Goldsmith, 1978) and published measurements of spectral sensitivity [e.g. electroretinogram (ERG) peaks of 565–570 nm (Kennedy & Bruno, 1961) and intracellular recordings of 556 nm and 600–620 nm (Nosaki, 1969) and 538–634 nm (Waterman & Fernandez, 1970)]. Woodcock & Goldsmith (1970) suggested that sensitivity maxima in the orange and red were due to the non-alignment of the stimulating beam with the optical axis of the cell, thereby allowing light to filter obliquely through red and brown proximal screening pigment surrounding the rhabdom. Goldsmith (1978) showed (theoretically) that the red-brown proximal screening pigments of crayfish and lobster are indeed capable of shifting the effective absorption of the rhabdoms enough to account for the discrepancy between the absorption peak of the visual pigment and the published measurements of spectral sensitivity. However, the effect on spectral sensitivity of the migration of the proximal screening pigment has not been documented.

49] Low-temperature spectrophotometry of intermediates of rhodopsin

Publisher Summary This chapter discusses the low-temperature spectrophotometry of intermediates of rhodopsin. It is well known that the absorption spectrum of rhodopsin in the visible range is in good agreement with the scotopic luminosity curve. As it takes a few milliseconds to generate the receptor potential of the visual cell after capture of a photon, the photoreaction of rhodopsin in less than a few milliseconds is important for elucidating the function of rhodopsin in visual transduction. Because such a fast photoreaction was so difficult to study at room temperature, most investigations had been carried out at low temperatures, where the thermal reaction rate of intermediates of rhodopsin is remarkably reduced. The application of low-temperature spectrophotometry to the rhodopsin system has led to discovery of several intermediates in the process of photobleaching of rhodopsin.

19] Purification of rat retinal rod outer segment membranes

This chapter discusses the purification of rat retinal rod outer segment (ROS) membranes. The retinal ROS membrane is a highly specialized unit capable of detecting a single photon in visual transduction. ROS preparations obtained from bovine and frog retinas are widely described and used. Rats are easily reared under controlled conditions of diet and environmental light, important variables for the visual system. To maintain rhodopsin in its dark-adapted state within the ROS membrane, it is necessary to perform the entire preparation procedure under dim red light. When ROS rhodopsin is maintained fully dark adapted in this way, the final purity and total yield obtained from the preparations are easily estimated using rhodopsin's absorption spectrum. The initial portion of this method is based on an extensive modification of the procedure described by Paper-master and Dreyer. The purity and yield of ROS membranes that have been harvested from either or both of bands one or two can be rapidly estimated spectrophotometrically.