Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

Specific photoisomerization of retinal in squid rhodopsin and metarhodopsin

The composition of retinal isomers in the photosteady-state mixtures formed from squid rhodopsin and metarhodopsin was determined by high-pressure liquid chromatography. A large amount of 9-cis-retinal was obtained at liquid N₂ temperature when rhodopsin was irradiated with orange light, but only small quantities of 9-cis-retinal were obtained at 15°C. Scarcely any 9-cis-retinal was produced from metarhodopsin by irradiation at liquid N₂ temperature. A large quantity of 7-cis-retinal was found in the photoproduct of rhodopsin irradiated at solid carbon dioxide temperature, but not at 15°C and liquid N₂ temperature. 7-cis-Retinal was not produced from metarhodopsin at any temperatures. These results indicate that the photoisomerization of retinal is regulated by the structure of the retinal-binding site of this protein. The formation of 9-cis- and 7-cis-retinals is forbidden in the metarhodopsin protein.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa: COMPARISON OF S186W AND D190N RHODOPSIN MUTANTS

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor''s active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin''s thermal stability and RP progression in patients.     

Key enzymes of the retinoid (visual) cycle in vertebrate retina

A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

A Ligand Channel through the G Protein Coupled Receptor Opsin

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 Å and is between 11.6 and 3.2 Å wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal β-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90° elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B.     

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10⁴ to 10⁵ free opsin molecules in a dark-adapted rod cell—a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.     

Retinobenzaldehydes as proper-trafficking inducers of folding-defective P23H rhodopsin mutant responsible for Retinitis Pigmentosa

The Retinitis pigmentosa (RP)-causing mutant of rhodopsin, P23H rhodopsin, is folding-defective and unable to traffic beyond the endoplasmic reticulum (ER). This ER retention, and in some cases aggregation, are proposed to result in ER-stress and eventually cell death. The endogenous rhodopsin ligand 11-cis-retinal and its isomer 9-cis-retinal have been shown to act as pharmacological chaperones, promoting proper folding and trafficking of the P23H rhodopsin. In spite of this promising effect, the development of retinals and related polyenealdehydes as pharmacological agents has been hampered by their undesirable properties, which include chemical instability, photolability, and potential retinoidal actions. Here, we report the design and synthesis of a class of more stable nonpolyene-type rhodopsin ligands, structurally distinct from, and with lower toxicity than, retinals. A structure–activity relationship study was conducted using cell-surface expression assay to quantify folding/trafficking efficiency of P23H rhodopsin.     

Molecular Mechanisms of Rhodopsin Retinitis Pigmentosa and the Efficacy of Pharmacological Rescue

Variants of rhodopsin, a complex of 11-cis retinal and opsin, cause retinitis pigmentosa (RP), a degenerative disease of the retina. Trafficking defects due to rhodopsin misfolding have been proposed as the most likely basis of the disease, but other potentially overlapping mechanisms may also apply. Pharmacological therapies for RP must target the major disease mechanism and contend with overlap, if it occurs. To this end, we have explored the molecular basis of rhodopsin RP in the context of pharmacological rescue with 11-cis retinal. Stable inducible cell lines were constructed to express wild-type opsin; the pathogenic variants T4R, T17M, P23A, P23H, P23L, and C110Y; or the nonpathogenic variants F220L and A299S. Pharmacological rescue was measured as the fold increase in rhodopsin or opsin levels upon addition of 11-cis retinal during opsin expression. Only Pro23 and T17M variants were rescued significantly. C110Y opsin was produced at low levels and did not yield rhodopsin, whereas the T4R, F220L, and A299S proteins reached near-wild-type levels and changed little with 11-cis retinal. All of the mutant rhodopsins exhibited misfolding, which increased over a broad range in the order F220L, A299S, T4R, T17M, P23A, P23H, P23L, as determined by decreased thermal stability in the dark and increased hydroxylamine sensitivity. Pharmacological rescue increased as misfolding decreased, but was limited for the least misfolded variants. Significantly, pathogenic variants also showed abnormal photobleaching behavior, including an increased ratio of metarhodopsin-I-like species to metarhodopsin-II-like species and aberrant photoproduct accumulation with prolonged illumination. These results, combined with an analysis of published biochemical and clinical studies, suggest that many rhodopsin variants cause disease by affecting both biosynthesis and photoactivity. We conclude that pharmacological rescue is promising as a broadly effective therapy for rhodopsin RP, particularly if implemented in a way that minimizes the photoactivity of the mutant proteins.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.     

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.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate retina responsible for continuous generation of 11-cis-retinal from its all-trans-isomer (1-3). Because 11-cis-retinal is the chromophore of rhodopsin and cone visual pigments (4), disabling mutations in genes encoding proteins of the retinoid cycle can cause a spectrum of retinal diseases affecting sight (3). Moreover, the efficiency of the mammalian visual system and health of photoreceptors and retinal pigment epithelium (RPE)² decrease significantly with age. Even in the presence of a functional retinoid cycle, A2E, retinal dimer (RALdi), and other toxic all-trans-retinal condensation products (5-7) can accumulate as a consequence of aging (8). Under experimental conditions, these compounds can produce toxic effects on RPE cells (9-11). Patients affected by age-related macular degeneration, Stargardt disease, or other retinal diseases associated with accumulation of surrogate markers, such as A2E, all develop retinal degeneration (12). Thus, elucidating the fundamental causes of these age-dependent changes is of increasing importance. Encouragingly, our understanding of both retinoid metabolism outside the eye and production of 11-cis-retinal unique to the eye has accelerated recently (Scheme 1) (1-3), and genetic mouse models are readily available to study these processes and their potential aberrations in vivo (13). Thus, a central question can be addressed, namely what initiates the death of photoreceptor cells and the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual cycle. After diffusion from the RPE, the visual chromophore, 11-cis-retinal, combines with rhodopsin and then is photoisomerized to all-trans-retinal. Most of the all-trans-retinal dissociates from opsin into the cytoplasm, where it is reduced to all-trans-retinol by RDHs, including RDH8. The fraction of all-trans-retinal that dissociates into the disc lumen is transported by ABCA4 into the cytoplasm (23) before it is reduced. All-trans-retinol then is translocated to the RPE, esterified by LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are associated with human macular degeneration, Stargardt disease, and age-related macular degeneration (55, 56).Several mechanisms associated with retinoid metabolism may contribute to different retinopathies (1). For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or chromophore in retinoid isomerase knock-out (Rpe65^-/-) mice leads to rapid degeneration of cone photoreceptors and slowly progressive death of rods (14). Such mice do not produce toxic condensation products from all-trans-retinal. Instead, their retinopathies have been attributed to continuous activation of visual phototransduction (15) due to either the basal activity of opsin (16-18) or disordered vectorial transport of cone visual pigments without bound chromophore (19). Paradoxically, an abnormally high flux of retinoids through the retinoid cycle can also lead to retinopathy in other mouse models (20, 21). Animal models featuring anomalies in the retinoid cycle illustrate the importance of chromophore regeneration and provide an approach to elucidating mechanisms involved in human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8 (retinol dehydrogenase 8), one of the main enzymes that reduces all-trans-retinal in rod and cone outer segments (22), and Abca4 (ATP-binding cassette transporter 4), which transports all-trans-retinal from the inside to the outside of disc membranes (23), rapidly accumulate all-trans-retinal condensation products and exhibit accentuated RPE/photoreceptor dystrophy at an early age (24). Although these studies suggest retinoid toxicity, it is still unclear if the elevated levels of retinal and/or its condensation products, such as A2E, are the cause of this retinopathy or merely a nonspecific reflection of impaired retinoid metabolism. Here, we report that spent chromophore, all-trans-retinal, is most likely responsible for photoreceptor degeneration in Rdh8^-/-Abca4^-/- mice. Toxic effects of all-trans-retinal include caspase activation and mitochondria-associated cell death.     

Retinal-protein interactions in bacteriorhodopsin monomers,dispersed in the detergent L-1690

Bacteriorhodopsin monomer dispersed in a solution of the detergent L-1690 could maintain the specific interaction between retinal and protein in the pH range 9.0-0.0 at 25°C. λ_max of the absorbance spectrum was 550 nm at pH 9.0, 556 nm at pH 5.5, 609 nm at pH 2.1 and 570 nm at pH 0.0. Increasing the NaCl concentration in the solution promoted formation of the 609 nm product at pH 5.0-3.0 and also its transition to the 570 nm product at pH 2.5-1.0. Retinal isomer analysis gave a ratio of 13-cis- to all-trans-retinal of 53 : 47 at pH 5.5. When the pH of the solution was reduced, the relative content of all-trans-retinal increased and the ratio of 13-cis- to all-trans-retinal was 14 : 86 at pH 0.0. Illumination of the solution at pH 7.2 yielded a product containing 9-cis-retinal or 9-cis, 13-cis-retinal, which may be due to a reaction other than the photoreaction cycle.     

Enzymatic reduction of 11-cis-retinal bound to cellular retinal-binding protein

Cellular retinal-binding protein from bovine retina purifies with bound 11-cis-retinal and 11-cis-retinol as endogenous ligands. Inasmuch as these retinoids are interconvertible by a dehydrogenase reaction the accessibility of the aldehyde function of bound 11-cis-retinal to chemical and enzymatic reducing agents was determined. An 11-cis-retinol dehydrogenase from retinal pigment epithelial microsomes, first described by Lion, F., Rotmans, J.P., Daemen, F.J.M. and Bonting, S.L. (Biochim. Biophys. Acta 384, 283–292, 1975) was found to reduce complexed 11-cis-retinal at pH 5.5 and 37°C rapidaly and nearly quantitatively. The product of the reduction, 11-cis-retinol, remained complexed with the binding protein following the reaction. Reduction proceeded 3-times more rapidly with NADH than with NADPH. No change in geometrical isomeric configuration occurred during the reaction. The dehydrogenase from retinal pigment epithelium oxidized 11-cis-retinol complexed with cellular retinal-binding protein at pH 8.5 in the presence of NAD. In spite of the ready enzymatic reduction of 11-cis-retinal complexed with cellular retinal-binding protein, the aldehyde function was inaccessible to several chemical reducing agents. Incubation of the complex with NaBH₄ at pH 7.5 and NaCNBH₃ or borane dimethylamine at pH 5.5 did not result in reduction of 11-cis-retinal unless the complex had been exposed to white light, a treatment known to produce all-rans-retinal which has little affinity for the binding protein. Liver alcohol dehydrogenase produced only 10% reduction of 11-cis-retinal complexed with cellular retinal-binding protein in 15 min at 37°C when added in amounts which produced about 60% reduction of the uncomplexed retinoid. The results suggest that the interaction between the 11-cis-retinol dehydrogenase and the 11-cis-retinal complexed to cellular retinal-binding protein is a specific one of that the binding protein may function as a substrate carrier for a dehydrogenase.     

Studies on a Missing Reaction in the Visual Cycle

DEVELOPMENT in the biochemistry of vision during the past twenty-five years can be summarized by equations (1) and (2) in Fig. 1, which envisage¹ that 11-cis-retinal combines with the visual protein opsin in a dark reaction to form the photolabile complex rhodopsin, λ_{max 497 nm}. When rhodopsin absorbs light it stimulates, through a process whose mechanism is not understood, the transmission of impulses, which are responsible for the visual sensation, although much is known about the biochemical changes accompanying the absorption of light by rhodopsin. These changes culminate in the formation of all-trans-retinal (λ_{max 385 nm}) and opsin (equation (2), Fig. 1), through a number of intermediates² and for the completion of the cycle one needs a molecular process which may regenerate 11-cis-retinal from all-trans-retinal (equation (3), Fig. 1).     

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 photorhodopsin₅₇₀ with vibrationally-excited all-trans-retinal and rhodopsin₄₉₈ 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 photorhodopsin₅₇₀ and for rhodopsin₄₉₈, 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.     

Further characterization of the lipid-depleted bovine rhodopsin obtained by cholate-ammonium sulfate fractionation

The rhodopsin preparation obtained by the method of ammonium sulfate fractionation contained 3–6 mol phospholipid and about 18 mol cholate per mol rhodopsin. The purified rhodopsin had 74% helical structure and showed a visible CD spectrum different from that of rhodopsin in the membrane. The rhodopsin was stable below but denatured gradually above 20°C. The lifetime of metarhodopsin I was long in this preparation. Regeneration capacity was low and only 30% of the original rhodopsin was regenerable by addition of 11-cis-retinal after bleaching.50 mol of phosphatidylcholine were maximally bound to 1 mol rhodopsin when the purified rhodopsin was mixed with phosphatidylcholine in 0.5% cholate. The rhodopsin recombined with lipid had properties similar to those of the original rhodopsin in the membrane. Exchange of cholate for other detergents was easily performed by dialysis. The rhodopsin preparation in which cholate was exchanged for digitonin gave almost the same CD, thermal stability and regenerability as those of a native rhodopsin in the membrane but metarhodopsin I still retained its long lifetime.     

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5^−/−Rdh11^−/− mice as compared with Rdh5^−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5^−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.