9, 13-dicis-Rhodopsin and its one-photon-one-double-bond isomerization

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh 6.10 , in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh 6.10 by UV-visible and FTIR spectroscopy and have revealed that Rh 6.10 is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C 13 ‫؍‬C 14 double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.Rhodopsin is the light receptor molecule in vertebrate rod photoreceptor cells and is involved in vision under dim light conditions. It senses light by its covalently bound chromophore, 11-cis retinal, which undergoes a light-dependent cis to alltrans isomerization to trigger conformational changes of the receptor leading to formation of the activated Meta II state and ultimately to an excitation of the photoreceptor cell (1, 2).In an attempt to create a light-stable rhodopsin, 11-cis-"locked" analogues of retinal were synthesized; the C 13 methyl group was bridged to C 10 of the polyene of retinal to prevent the activating isomerization around the C 11 ϭC 12 double bond. Several different bridges were tested, leading to the synthesis of 11-cis-locked retinals with 5-, 6-, 7-, 8-, and even 9-member ring structures (Ref. 3 and references in Ref. 4). Much attention had been drawn to 11-cis ring-constrained retinals with a 6-member ring, Re 6.10 , as they recombined with opsin to pigments that were capable of at least some rudimentary photochemistry and some marginal activity toward the visual G protein, transducin (5, 6). Re 6.10 exists in four isomeric states, which can be grouped as 9-trans and 9-cis, depending on the isomeric state of the C 9 double bond (Scheme 1). A recent study characterized the overall shape of these four isomers by their affinities for either 11-cis-or all-trans-retinol dehydrogenase (7). ...

Section: Discussionmentioning

confidence: 96%

mentioning

confidence: 99%

A Nonbleachable Rhodopsin Analogue with a Slow Photocycle

Vogel

Fan

Lüdeke

et al. 2002

“…For those experiments the pigment was solubilized in detergent, which has a dramatic effect both on the Meta I/Meta II equilibrium of native rhodopsin (45) and on the flexibility of the chromophore binding pocket to accommodate different isomers (46).…”

Section: Discussionmentioning

confidence: 99%

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

Fan

Siebert

Sheves

et al. 2002

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.Rhodopsin, a seven-transmembrane helical protein, is composed of 348 amino acids and a ligand, 11-cis retinal, which covalently binds to the protein through a protonated Schiff base linkage to the ⑀-amino group of Lys-296 in the center of helix 7 (1). Glu-113 at helix 3 serves as the counterion of the protonated Schiff base (2-4). The recently resolved crystal structure of rhodopsin (5) provides detailed structural information on the interactions between the retinal chromophore and its surrounding residues, which contribute to the red-shifted absorption maximum of the chromophore ( max 500 nm) relative to protonated retinal Schiff base in methanol solution ( max 440 nm) and to the high pK a of the protonated Schiff base (6). Following light absorption, the retinal chromophore isomerizes from 11-cis to trans configuration in 200 fs (7). A series of photointermediates are produced that can be trapped below characteristic transition temperatures (8, 9): bathorhodopsin ( max 543 nm, T Ͻ Ϫ140°C), lumirhodopsin ( max 497 nm, T Ͻ Ϫ40°C), and metarhodopsin I (Meta I, 1 max 478 nm) above Ϫ40°C, which equilibrates with metarhodopsin II (Meta II, max 380 nm). Meta II is the active species, capable of activating transducin, the visual G protein. The equilibrium between Meta I and Meta II is affected by temperature, pressure, pH, and glycerol (9), as well as by ions (10, 11).Meta II formation is associated with a movement of helix 6 relative to helix 3 (12, 13), a translocation of a proton from the Schiff base to its counterion, Glu-113, and re...

“…Briefly, the retinaloximes of lightirradiated (Y52 cutoff filter; Toshiba) and non-irradiated purified samples were extracted, and HPLC analysis was performed (20,21). Retinal composition was calculated from the area of the peaks and the absorption coefficients previously reported (22).…”

Section: Preparation Of the Alkyl-retinal Schiff Bases-retinylidenementioning

confidence: 99%

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Matsuyama

Yamashita

Imai

et al. 2010

Self Cite

Rhodopsin is an extensively studied member of the G proteincoupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.Rhodopsin is the photoreceptor molecule present in vertebrate eyes, which senses light stimuli and initiates a signaling cascade mediated by the G protein. Rhodopsin is a member of the family-A G protein-coupled receptors (GPCRs), 3 and therefore it is presumed to have diverged from a diffusible ligand binding GPCR (1). However, in contrast with other GPCRs where diffusible ligands interact with their receptors strictly by noncovalent interactions, the binding of the retinal to opsin involves a covalent bond between the receptor and the ligand. Rhodopsin has the 11-cis-retinal chromophore covalently bound in its protein moiety acting as an inverse agonist in the dark, and light absorption causes the generation of the agonist all-trans-retinal through cis-trans photoisomerization of the chromophore (2).The covalent bond is formed between the aldehyde group of the retinal and the ⑀-amino group of lysine 296 4 located at the transmembrane helix VII of rhodopsin, through a protonated Schiff base linkage (3, 4). Formation of the protonated Schiff base is an important structural feature for photoreception, because the protonation of the Schiff base causes electron delocalization of the chromophore, resulting in a red shift of the absorption spectrum of rhodopsin, which allows it to absorb visible light (5). Moreover, the positive charge of Lys-296 and the negative charge of Glu-113 form an ionic pair, thus forming an intramolecular switch known as the opsin lock, which locks the receptor in an inactive state (6, 7). This ionic interaction is maintained in ...