Rhodopsin Analogs Research Articles

Isorhodopsin: An Undervalued Visual Pigment Analog

Rhodopsin, the first visual pigment identified in the animal retina, was shown to be a photosensitive membrane protein containing covalently bound retinal in the 11-cis configuration, as a chromophore. Upon photoexcitation the chromophore isomerizes in femtoseconds to all-trans, which drives the protein into the active state. Soon thereafter, another geometric isomer—9-cis retinal—was also shown to stably incorporate into the binding pocket, generating a slightly blue-shifted photosensitive protein. This pigment, coined isorhodopsin, was less photosensitive, but could also reach the active state. However, 9-cis retinal was not detected as a chromophore in any of the many animal visual pigments studied, and isorhodopsin was passed over as an exotic and little-relevant rhodopsin analog. Consequently, few in-depth studies of its photochemistry and activation mechanism have been performed. In this review, we aim to illustrate that it is unfortunate that isorhodopsin has received little attention in the visual research and literature. Elementary differences in photoexcitation of rhodopsin and isorhodopsin have already been reported. Further in-depth studies of the photochemical properties and pathways of isorhodopsin would be quite enlightening for the initial steps in vision, as well as being beneficial for biotechnological applications of retinal proteins.

Impacts of retinal polyene (de)methylation on the photoisomerization mechanism and photon energy storage of rhodopsin.

Ab initio multiconfigurational quantum chemical methodology combined with molecular mechanics (CASPT2//CASSCF/AMBER) was applied to probe impacts of retinal protonated Schiff base (RPSB) polyene methylation and/or demethylation on the mechanism of photochemical isomerization in bovine rhodopsin. We have examined structural and spectroscopic properties of wild-type rhodopsin (with 11-cis-9,13-dimethyl-RPSB) and artificial rhodopsins, hosting four 11-cis-RPSB derivatives, 13-demethyl-, 9-demethyl-, 10-methyl-13-demethyl-, and 10-methyl-RPSB, evolving along the photoisomerization coordinate. It is found that the addition of 10-methyl or/and deletion of 9-/13-methyl groups do not appear to interfere structurally with the photoisomerization pathway in the S1 excited state. Remarkably, the two-mode space-saving mechanism initiated by bond order inversion and followed by asynchronous bicycle-pedal distortion in the RPSB backbone drives the photoreaction in all rhodopsin analogues studied here. However, methylation and/or demethylation is responsible for perturbation of excess energy deposited in the conical intersection structures. The analysis of photon energy stored by bathorhodopsin in synthetic pigments reveals that it is affected by steric crowding of methyl substituents in the RPSB backbone.

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

To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. 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 obtained by extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-((3)H)-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-(32)P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 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 light-dependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. 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.

Photochemistry of Visual Pigment Chromophore Models by Ab Initio Molecular Dynamics

Ab initio excited-state molecular dynamics calculations have been performed to study the effect of methyl substitution and chromophore distortion on the photoreaction of different four-double-bond retinal model chromophores. Randomly distributed starting geometries were generated by zero-point energy sampling; after Franck-Condon excitation the reaction was followed on the S1 surface. For determining the photoproduct and its configuration, a simplified approach--torsion angle following--is discussed and applied. We find that chromophore distortion significantly affects the outcome of the photoreaction: with dihedral angles taken from the rhodopsin-embedded 11-cis-retinal chromophore, the reaction rate of the model chromophore is increased by a factor of 3 compared to that of the relaxed chromophore. Also, the reaction proceeds in a completely stereoselective manner involving only the cis double bond and with a minimum quantum yield of 72%. Bond torsion is more effective than methyl substitution for fast and selective photochemistry, which is in agreement with photophysical measurements on rhodopsin analogues. We conclude that apart from the geometric distortions caused by the protein pocket it is not necessary to postulate other specific interactions between the protein and the chromophore to effect the selective and ultrafast photoreaction in rhodopsin.

Methyl Substituents at the 11 or 12 Position of Retinal Profoundly and Differentially Affect Photochemistry and Signalling Activity of Rhodopsin

The C-11 = C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11 = C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10–C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 °C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin.

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.

Primary Photoreaction Processes in Vision.

This article reviews the primary reaction processes in rhodopsin, a light-sensor protein in our vision. Rhodopsin has an 11-cis retinal as the chromophore, which binds covalently with a lysine residue through a protonated Schiff base linkage. 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 fs 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.

Rhodopsin with 11-cis-Locked Chromophore Is Capable of Forming an Active State Photoproduct

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.

Isomers of 10-fluoro-20- 13C-retinal and the corresponding rhodopsin analogs

The doubly-labeled 10-fluoro-20- 13C-retinal has been prepared. Five isomers have been isolated and two isomeric rhodopsin analogs prepared. The latter are intended for REDOR distance measurements.

Retinoids and related compounds. Part 26. Synthesis of (11Z)-8,18-propano- and methano-retinals and conformational study of the rhodopsin chromophore†

In order to clarify the conformation of the chromophore in rhodopsin, especially the effect of the torsional angle around the C6–C7 single bond on the CD spectrum, 8,18-propano- and methano-retinal 4 and 5 were prepared via the palladium-catalyzed coupling reaction of vinyl triflates derived from bicyclic ketones 10 and 27 with methyl (E)-3-(trimethylstannyl)but-2-enoate. In a binding experiment with bovine opsin, retinal analogs 4 and 5 afforded the new rhodopsin analogs. Although the opsin shifts of these pigments were similar to that of the native rhodopsin, CD spectra exhibited the characteristic feature owing to the locked structures of retinal analogs. This fact strongly indicates that the CD spectra were significantly influenced by the torsional angles around the 6–7 and 8–9 single bonds of the chromophore in the protein.

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.

An Additional Methyl Group at the 10-Position of Retinal Dramatically Slows down the Kinetics of the Rhodopsin Photocascade

The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.

Retinoids and related compounds. Part 20.1 Synthesis of (11Z)-8,18-ethanoretinal and a conformational study of the rhodopsin chromophore

A. Wada, M. Sakai, Y. Imamoto, Y. Shichida, M. Yamauchi and M. Ito, J. Chem. Soc., Perkin Trans. 1, 1997, 1773 DOI: 10.1039/A700796E

Structure around C6-C7 bond of the chromophore in bathorhodopsin: low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs.

To elucidate the structural changes near the beta-ionone ring region of the chromophore during the photobleaching process of rhodopsin, the photochemical and subsequent thermal reactions of rhodopsin analogs, whose retinylidene chromophores were fixed in a 6s-cis form with a five-membered ring (6,5-rhodopsin) and a seven-membered ring (6,7-rhodopsin), respectively, were investigated by low-temperature spectroscopy. Like rhodopsin, both the rhodopsin analogs convert to the respective batho-intermediates upon absorption of light at -190 degrees C. The extinction coefficient of batho-intermediate of 6,5-rhodopsin is similar to that of bathorhodopsin, while that of 6,7-rhodopsin is considerably smaller than that of bathorhodopsin. Like bathorhodopsin, the batho-intermediate of 6,5-rhodopsin directly converts to lumi-intermediate, while that of 6,7-rhodopsin first converts to a blue-shifted intermediate and then to lumi-intermediate. These results strongly suggest that the structure around the beta-ionone ring region of the bathorhodopsin chromophore resembles 6,5-retinal rather than 6,7-retinal. From the comparison of the structural features among retinal, 6,5-retinal, and 6,7-retinal, a possible conformation around C6-C7 bond of the bathorhodopsin chromophore is discussed.

Real-Time Detection of 60-fs Isomerization in a Rhodopsin Analog Containing Eight-Membered-Ring Retinal

Femtosecond fluorescence spectroscopy has been used to investigate the excited-state dynamics of a rhodopsin analog having retinal modified by a flexible eight-membered ring. The fluorescence lifetime was determined to be 60 fs throughout the observed wavelengths. Combined with the previous transient absorption results (Mizukami, T.; Kandori, H.; Shichida, Y.; Chen, A.-H.; Derguini, F.; Caldwell, C. G.; Bigge, C. F.; Nakanishi, K.; Yoshizawa, T. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 4072-4076), the ultrafast process was interpreted in terms of rapid deformation around the C11−C12 double bond, i.e., isomerization, occurring as fast as vibrational motions of the chromophore. The ultrafast reaction suggests that the isomerization occurs in a coherent manner. On the basis of the present results for the modified rhodopsin, the primary processes in the native rhodopsin are discussed.

A 19F NMR Study of Rhodopsin Analogs: Use of Vinylfluororetinal Chromophores

19F NMR spectra of 11-cis and 9-cis isomers of six fluorinated rhodopsin analogs with the label(s) located at the vinylic positions of the polyene chain (8-F, 10-F, 12-F, 14-F, 8,12-F2, 10,14-F2) are reported along with their UV−vis and CD spectra. The regiospecific F chemical shift data are analyzed in terms of chromophore changes and local perturbation resulting from specific interactions with protein. Two analogs (11-cis-12-F and 11-cis-8-F) and also 9,11-di-cis-12-F possess FOS (fluorine opsin shift) values uniquely different from others. Ab initio F NMR chemical shielding calculations of model structures provide support that a strong protein perturbation to the 12-F position prevails in the binding cavity and that F-8 shift is sensitive to variation of the nearby dihedral angle(s). Possible causes for the broad line width of the F signals of these membrane proteins are discussed.

Rhodopsins containing 6- to 9-membered rings. The triggering process of visual transduction

Abstract

Synthesis of retinals with eight- and nine-membered rings in the side chain. Models for rhodopsin photobleaching intermediates

The triggering process of rhodopsin activation leading to visual transduction has remained a key problem in the chemistry of vision. In order to provide probes to explore this process, retinal analogs ret8 3 and ret9 4, with the 11-ene cis-locked by 8- and 9-membered rings, have been synthesized. These could lead to pigment analogs which mightaccomodate a transoid chromophore upon irradiation, thus providing unique models for rhodopsin activation studies. Enzyme asays and flash photolysis of rhodopsin analogs incorporating ret8 and other retinal analogs showed that the triggering process requires complete 11-cis to trans isomerization involving the entire polyene moiety. The syntheses of ret8 3 and ret9 4 double bond isomers are described

Photoisomerization mechanism of the rhodopsin chromophore: picosecond photolysis of pigment containing 11-cis-locked eight-membered ring retinal.

The primary photochemical event in rhodopsin is an 11-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal 11-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked 11-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo- and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the 11-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.

A new rhodopsin analog involving 11Z-8,18-ethanoretinal as a chromophore.

A new rhodopsin analog involving 11Z-8,18-ethanoretinal as a chromophore.