Photoreversal of Rhodopsin Bleaching

A mechanistic scheme, showing certain steps of rhodopsin bleaching, provides two ways of viewing the bleaching process: (a) The rate of bleaching depends upon the net rate of accumulation of labile species; and (b) the number of labile molecules which accumulates in a certain period is the number which has absorbed an odd number of quanta by the end of that period. Both views, based on the photoreversibility of bleaching, lend themselves to concise mathematical formulation. The expected amounts of bleaching at various intensities, calculated according to these formulae, give very close fits to the experimental data. The relevance of these results to other experiments is pointed out and emphasis is placed on the explanation of observed quantum efficiencies which obtain at both low and high intensities.     

A Spectroscopic Study of Rhodopsin Alpha-Helix Orientation

Polarized Fourier transform infrared spectroscopy and far ultraviolet circular dichroism of oriented multilamellar films of photoreceptor membranes indicate rhodopsin alpha-helices are predominantly oriented perpendicular to the bilayer plane.     

The Orientation of Rhodopsin and Other Pigments in Dry Films

When rhodopsin in a gelatin film is dried, the rhodopsin chromophores orient primarily in the plane of the film. When the film is wetted, the chromophores disorient. These changes are reversible. When rhodopsin in a wet film. is bleached in the presence of hydroxylamine and redried, the retinal oxime which results is oriented more perpendicularly to the plane of the film. These orientations in dry gelatin films resemble those in the disc membranes of rod outer segments. A variety of other proteins are similarly oriented in dry gelatin films: methemoglobin, cytochrome c, phycocyanin. Films of methemoglobin and cytochrome c display prominently the high Soret band near 410 nm when measured with unpolarized light passing through the face of the fim, but display no Soret band at all with light passing through the edge of the film. All of these orientations imply a large asymmetry of the protein micelles, perhaps conferred upon them by linear polymerization in the course of drying. Such asymmetry can be demonstrated directly with rhodopsin. A wet paste of rhodopsin-digitonin micelles, sheared between glass slides, becomes highly oriented, the rhodopsin chromophores lining up in the direction of shear, the retinal oxime produced by bleaching orienting more perpendicularly to the shear.     

Photoisomerization in Rhodopsin

This article reviews the primary reaction processes in rhodopsin, a photoreceptive pigment for twilight vision. Rhodopsin has an 11-cis retinal as the chromophore, which binds covalently with a lysine residue through a protonated Schiff base linkage. Absorption of a photon by rhodopsin initiates the primary photochemical reaction in the chromophore. Picosecond time-resolved spectroscopy of 11-cis locked rhodopsin analogs revealed that the cis-trans isomerization of the chromophore is the primary reaction in rhodopsin. Then, generation of femtosecond laser pulses in the 1990s made it possible to follow the process of isomerization in real time. Formation of photorhodopsin within 200 fsec was observed by a transient absorption (pump–probe) experiment, which also revealed that the photoisomerization in rhodopsin is a vibrationally coherent process. Femtosecond fluorescence spectroscopy directly captured excited-state dynamics of rhodopsin, so that both coherent reaction process and unreacted excited state were observed. Faster photoreaction of the chromophore in rhodopsin than that in solution implies that the protein environment facilitates the efficient isomerization process. Such contributions of the protein residues have been monitored by infrared spectroscopy of rhodopsin, bathorhodopsin, and isorhodopsin (9-cis rhodopsin) at low temperatures. The crystal structure of bovine rhodopsin recently reported will lead to better understanding of the mechanism in future.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

Rhodopsin kinetics in the cat retina

The bleaching and regeneration of rhodopsin in the living cat retina was studied by means of fundus reflectometry. Bleaching was effected by continuous light exposures of 1 min or 20 min, and the changes in retinal absorbance were measured at 29 wavelengths. For all of the conditions studied (fractional bleaches of from 65 to 100%), the regeneration of rhodopsin to its prebleach levels required greater than 60 min in darkness. After the 1-min exposures, the difference spectra recorded during the first 10 min of dark adaptation were dominated by photoproduct absorption, and rhodopsin regeneration kinetics were obscured by these intermediate processes. Extending the bleaching duration to 20 min gave the products of photolysis an opportunity to dissipate, and it was possible to follow the regenerative process over its full time-course. It was not possible, however, to fit these data with the simple exponential function predicted by first-order reaction kinetics. Other possible mechanisms were considered and are presented in the text. Nevertheless, the kinetics of regeneration compared favorably with the temporal changes in log sensitivity determined electrophysiologically by other investigators. Based on the bleaching curve for cat rhodopsin, the photosensitivity was determined and found to approximate closely the value obtained for human rhodopsin; i.e., the energy Ec required to bleach 1-e-1 of the available rhodopsin was 7.09 log scotopic troland-seconds (corrected for the optics of the cat eye), as compared with approximately 7.0 in man.     

Rhodopsin of the Larval Mosquito

Larvae of the mosquito Aedes aegypti have a cluster of four ocelli on each side of the head. The visual pigment of each ocellus of mosquitoes reared in darkness was characterized by microspectrophotometry, and found to be the same. Larval mosquito rhodopsin (λ_max = 515 nm) upon short irradiation bleaches to a stable photoequilibrium with metarhodopsin (λ_max = 480 nm). On long irradiation of glutaraldehyde-fixed tissues or in the presence of potassium borohydride, bleaching goes further, and potassium borohydride reduces the product, retinal, to retinol (vitamin A₁). In the presence of hydroxylamine, the rhodopsin bleaches rapidly, with conversion of the chromophore to retinaldehyde oxime (λ_max about 365 nm).     

Stability of Rhodopsin in Detergent Solutions

The thermal stability of lipid-free rhodopsin in solutions of a homologous series of alkyltrimethylammonium bromide detergents and one nonionic detergent, dodecyl-β-maltoside, has been studied as a function of detergent concentration. Rhodopsin thermal stability increases with increasing chain length within the homologous series of ionic detergents, and for chain lengths greater than 10 carbon atoms increases with increasing detergent concentration up to a “critical” concentration that depends on the chain length. Stability also increases with increasing detergent concentration for rhodopsin in solutions of the nonionic detergent. These results may be rationalized in terms of the dependence of micelle packing density on the detergent chain length, head group, and concentration.     

Rhodopsin Rotates in the Visual Receptor Membrane

Dichroism can be photoinduced in a frog retina once it has been fixed with glutaraldehyde. This dichroism is absent in the normal retina because rhodopsin is free to undergo rotational Brownian motion.     

Methylation of Bovine Rhodopsin

METHYLATED basic amino-acids have been detected in many proteins¹ and we report here the methylation of rhodopsin^2–5. Bovine rhodopsin has been found to contain MML (?-N-monomethyl lysine), DML (?-N-dimethyl lysine) and 3-MH (3-methyl histidine). Residues of TML (?-N-trimethyl lysine) and DMA (dimethyl arginine) were also detected in an un-characterized protein association with rhodopsin which is solubilized from outer segments by 1 % nonionic detergent ‘Ammonyx LO’. In vitro methylation of the basic residues in bovine rhodopsin was also detected after dissected retinas had been incubated in a nutrient medium containing ³H-(methyl) methionine.     

Dynamics of the Internal Water Molecules in Squid Rhodopsin

Understanding the mechanism of G-protein coupled receptors action is of major interest for drug design. The visual rhodopsin is the prototype structure for the family A of G-protein coupled receptors. Upon photoisomerization of the covalently bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G-protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The crystal structure of the visual rhodopsin from squid solved recently suggests that a chain of water molecules extending from the retinal toward the cytoplasmic side of the protein may play a role in the signal transduction from the all-trans retinal geometry to the activated receptor. As a first step toward understanding the role of water in rhodopsin function, we performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. The simulation indicates that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region and form a persistent hydrogen-bond network in connecting Y315 to W274 via D80.     

Molecular Weight of Frog Rhodopsin

RECENT studies have suggested that bovine^1–4, rat⁴ and frog^4,5 rhodopsins all have molecular weights close to 28,000. We have now determined the molecular weight of frog (Rana catesbeiana) rhodopsin using several different techniques and obtain a value for the maximum molecular weight of approximately 40,000. Our previous report⁵ was incorrect because of an error in the calibration of the amino-acid analyser used. These results, together with those of Cavanagh and Wald on cattle rhodopsin⁶, suggest that the lower values may be in error.     

Rhodopsin photoproducts in 2D crystals

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

Rotational Diffusion of Rhodopsin in the Visual Receptor Membrane

Transient photodichroism in the frog retina reveals that rhodopsin has a relaxation time of 20 µs. The site rhodopsin occupies in the membrane must therefore be highly fluid. This suggests rhodopsin may be a diffusional carrier.