EFFECT OF THE PHYSICAL STATE OF PHOSPHOLIPID ON RHODOPSIN REGENERATION FROM RETINYLIDENE PHOSPHOLIPID

Abstract— Rhodopsin regeneration in rod membranes involves reactions of all -trans retinal (released from bleached pigment) with phosphatidylethanolamine, photic isomerization of retinal, and binding of 11-cis retinal to opsin. This investigation demonstrated that formation of retinylidene phospholipid and retinal binding to opsin were both affected by the physical state of phospholipid. A fluid membraneous environment provided by the acyl chains of phospholipid was essential for these reactions to proceed efficiently. The retinal moiety of retinylidene phospholipid appeared to be directly transferred to opsin by transimination.     

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.     

Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.     

Recent bioorganic studies on rhodopsin and visual transduction

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.     

Activity switches of rhodopsin

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.     

The role of the beta-ionone ring in the photochemical reaction of rhodopsin

Time-dependent density functional theory (TDDFT) calculations on the photoabsorption process of the 11-cis retinal protonated Schiff base (PSB) chromophore show that the Franck-Condon relaxation of the first excited state of the chromophore involves a torsional twist motion of the beta-ionone ring relative to the conjugated retinyl chain. For the ground state, the beta-ionone ring and the retinyl chain of the free retinal PSB chromophore form a -40 degrees dihedral angle as compared to -94 degrees for the first excited state. The double bonds of the retinal are shorter for the fully optimized structure of the excited state than for the ground state suggesting a higher cis-trans isomerization barrier for the excited state than for the ground state. According to the present TDDFT calculations, the excitation of the retinal PSB chromophore does not primarily lead to a reaction along the cis-trans torsional coordinate at the C11-C12 bond. The activation of the isomerization center seems to occur at a later stage of the photo reaction. The results obtained at the TDDFT level are supported by second-order M?ller-Plesset (MP2) and approximate singles and doubles-coupled cluster (CC2) calculations on retinal chromophore models; the MP2 and CC2 calculations yield for them qualitatively the same ground state and excited-state structures as obtained in the density functional theory and TDDFT calculations.     

Rhodopsin deactivation is affected by mutations of Tyrl91

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyrl91 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyrl91, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyrl91 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photo-isomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.     

The opsin shift and mechanism of spectral tuning in rhodopsin

Molecular dynamics simulations and combined quantum mechanical and molecular mechanical calculations have been performed to investigate the mechanism of the opsin shift and spectral tuning in rhodopsin. A red shift of -980 cm(-1) was estimated in the transfer of the chromophore from methanol solution environment to the protonated Schiff base (PSB)-binding site of the opsin. The conformational change from a 6-s-cis-all-trans configuration in solution to the 6-s-cis-11-cis conformer contributes additional -200 cm(-1), and the remaining effects were attributed to dispersion interactions with the aromatic residues in the binding site. An opsin shift of 2100 cm(-1) was obtained, in reasonable accord with experiment (2730 cm(-1)). Dynamics simulations revealed that the 6-s-cis bond can occupy two main conformations for the β-ionone ring, resulting in a weighted average dihedral angle of about -50°, which may be compared with the experimental estimate of -28° from solid-state NMR and Raman data. We investigated a series of four single mutations, including E113D, A292S, T118A, and A269T, which are located near the PSB, along the polyene chain of retinal and close to the ionone ring. The computational results on absorption energy shift provided insights into the mechanism of spectral tuning, which involves all means of electronic structural effects, including the stabilization or destabilization of either the ground or the electronically excited state of the retinal PSB.     

RECONSTITUTION OF SQUID RHODOPSIN IN RHABDOMAL MEMBRANES

Abstract— Squid opsin which is capable of combining with 11- cis or 9- cis retinal to reconstitute photo-pigment has been prepared by irradiation of rhabdomal membranes with orange light (> 530 nm) in the presence of 0.2 M hydroxylamine. When the irradiation is carried out either at concentrations of hydroxylamine higher than 0.2 M or with light of wavelength shorter than 530 nm, rhodopsin in the membranes is bleached quickly, but the ability of the resultant opsin to form rhodopsin is greatly reduced.
The optimum pH for rhodopsin regeneration in rhabdomal membranes was found to be between 6.5 and 8.5. The rate of regeneration of rhodopsin increases with raising temperature, and at about 20°C it is almost the same as that of isorhodopsin. Even after solubilization in digitonin solution, opsin still preserves the ability to reform rhodopsin.
All- trans retinal can be incorporated into retinochrome-bearing membranes, in which it is isomerized into 11- cis isomer by the photoisomerase activity of retinochrome. Rhabdomal membranes retaining active opsin can take up 11- cis retinal from retinochrome membranes so as to synthesize rhodopsin.     

Novel retinylidene iminium salts for defining opsin shifts: synthesis and intrinsic chromophoric properties

Retinal Schiff bases serve as chromophores in many photoactive proteins that carry out functions such as signalling and light-induced ion translocation. The retinal Schiff base can be found as neutral or protonated, as all-trans, 11-cis or 13-cis isomers and can adopt different conformations in the protein binding pocket. Here we present the synthesis and characterisation of isomeric retinylidene iminium salts as mimics blocked towards isomerisation at the C11 position and conformationally restrained. The intrinsic chromophoric properties are elucidated by gas phase absorption studies. These studies reveal a small blue-shift in the S0-->S1 absorption for the 11-locked derivative as compared to the unlocked one. The gas phase absorption spectra of all the cationic mimics so far investigated show almost no absorption in the blue region. This observation stresses the importance of protein interactions for colour tuning, which allows the human eye to perceive blue light.     

SPIN LABELED CYSTEINES AS SENSORS FOR PROTEIN-LIPID INTERACTION AND CONFORMATION IN RHODOPSIN

In stoichiometric amounts, the spin label N-tempoyl-(p-chloromercuribenzamide) reacts rapidly with one cysteine residue in membrane-bound bovine rhodopsin. This residue is distinct from the two reactive cysteines previously used as attachment sites for spectroscopic labels, and is on the external surface of the protein near the cytoplasmic membrane/aqueous interface. The spin-labeled side chain has revealed a light-induced conformational change in membrane-bound rhodopsin that is apparently not associated with protein aggregation. The changes are reversible upon the addition of 11-cis retinal, and the magnitude of the change is dependent on the identity of the phospholipid in the surrounding bilayer. Alteration of lipid composition has a much larger effect on bleached rhodopsin than rhodopsin itself, indicating that the former is more readily deformable in response to changes in bilayer properties. This is consistent with the loss of 11-cis retinal binding energy in opsin compared to rhodopsin. These results provide direct structural evidence that the conformation of a membrane protein can be modulated by the lipid properties.     

Quantum yield of CHAPSO-solubilized rhodopsin and 3-hydroxy retinal containing bovine opsin.

The quantum yields of bleaching for two artificial pigments, bovine opsin combined with (3R)-3-hydroxy retinal or (3R,S)-3-methoxy retinal, were determined in comparison to the value for regenerated bovine rhodopsin. Regeneration of the visual pigments was performed by incubation of 3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate (CHAPSO)-solubilized opsin with the 11-cis isomers of retinal and the respective retinal derivatives. The extinction coefficients of the pigments in CHAPSO were determined to 35,000 M-1 cm-1 (native rhodopsin), 35,300 M-1 cm-1 (regenerated rhodopsin) and 34,500 M-1 cm-1 (3-OH retinal opsin). With respect to rhodopsin (lambda max: 500 nm), the pigments carrying the substituted chromophores exhibit blue shifted absorbance maxima (3-hydroxy and 3-methoxy retinal opsin: 488 nm). In parallel experiments under absolutely identical conditions we find related to the value of CHAPSO solubilized rhodopsin (identical to 1) a quantum efficiency of bleaching for the 3-hydroxy pigment of 1.2.     

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

All-trans-N-retinylidenetryptamine Schiff base in surfactant solubilized water pools in heptane—a fluorescence study

All-trns-N-retinylidenetryptamine Schiff base was incorporated into aerosol-OT (AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/heptane reverse micelles. This micellar system was used as a model to study the retinal-tryptophan interactions in retinal proteins. The retinylidene Schiff base remains stable in the presence of reverse micelle-solubilized water pools. Partition coefficient and microviscosity measurements show that the Schiff base is located in the micellar interphase. The results are discussed in terms of the interaction between the retinylidene chromophore and the active site environment of rhodopsin and bacteriorhodopsin. In the present model, the quencher and emitting units are covalently attached, and are separated by two carbon spacer units. The fluorescence emission data obtained for the micelle-intercalated Schiff base chromophore are compared with the fluorescence of the native protein and intermediates in the photochemical cycle of bacteriofhodopsin. A comparison of the data obtained for tryptamine and the Schiff base with the results available for bacteriorhodopsin and bacterioopsin reveals that there is a large degree of quenching on intercalation of the retinylidene chromophore in the vicinity of the fluorophore. Evidence provided by this model suggests that energy transfer to retinal can occur from tryptophan residues located in the retinal pocket in the native protein. Thus the retinylidene unit can act as a quencher of the energy of tryptophan, the nature and extent of which may depend on the conformation and relative orientation of the protein-bound fluorophore.     

Comparative Analysis of GPCR Crystal Structures†

The phototransduction cascade is perhaps the best understood model system for G protein‐coupled receptor (GPCR) signaling. Phototransduction links the absorption of a single photon of light to a decrease in cytosolic cGMP. Depletion of the cGMP pool induces closure of cGMP‐gated cation channels resulting in the hyperpolarization of photoreceptor cells and consequently a neuronal response. Many biochemical and both low‐ and high‐resolution structural approaches have been utilized to increase our understanding of rhodopsin, the key molecule of this signaling cascade. Rhodopsin, a member of the GPCR or seven‐transmembrane spanning receptor superfamily, is composed of a chromophore, 11‐cis‐retinal that is covalently bound by a protonated Schiff base linkage to the apo‐protein opsin at Lys²⁹⁶ (in bovine opsin). Upon absorption of a photon, isomerization of the chromophore to an all‐trans‐retinylidene conformation induces changes in the rhodopsin structure, ultimately converting it from an inactive to an activated state. This state allows it to activate the heterotrimeric G protein, transducin, by triggering nucleotide exchange. To fully understand the structural and functional aspects of rhodopsin it is necessary to critically examine crystal structures of its different photointermediates. In this review we summarize recent progress on the structure and activation of rhodopsin in the context of other GPCR structures.     

Structure, spectroscopy, and spectral tuning of the gas-phase retinal chromophore: the beta-ionone "handle" and alkyl group effect

The low-lying singlet states (i.e. S0, S1, and S2) of the chromophore of rhodopsin, the protonated Schiff base of 11-cis-retinal (PSB11), and of its all-trans photoproduct have been studied in isolated conditions by using ab initio multiconfigurational second-order perturbation theory. The computed spectroscopic features include the vertical excitation, the band origin, and the fluorescence maximum of both isomers. On the basis of the S0-->S1 vertical excitation, the gas-phase absorption maximum of PSB11 is predicted to be 545 nm (2.28 eV). Thus, the predicted absorption maximum appears to be closer to that of the rhodopsin pigment (2.48 eV) and considerably red-shifted with respect to that measured in solution (2.82 eV in methanol). In addition, the absorption maxima associated with the blue, green, and red cone visual pigments are tentatively rationalized in terms of the spectral changes computed for PSB11 structures featuring differently twisted beta-ionone rings. More specifically, a blue-shifted absorption maximum is explained in terms of a large twisting of the beta-ionone ring (with respect to the main conjugated chain) in the visual S-cone (blue) pigment chromophore. In contrast, the chromophore of the visual L-cone (red) pigment is expected to have a nearly coplanar beta-ionone ring yielding a six double bond fully conjugated framework. Finally, the M-cone (green) chromophore is expected to feature a twisting angle between 10 and 60 degrees. The spectroscopic effects of the alkyl substituents on the PSB11 spectroscopic properties have also been investigated. It is found that they have a not negligible stabilizing effect on the S1-S0 energy gap (and, thus, cause a red shift of the absorption maximum) only when the double bond of the beta-ionone ring conjugates significantly with the rest of the conjugated chain.     

Modulating rhodopsin receptor activation by altering the pKa of the retinal Schiff base

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pKa of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal beta-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.     

Iodopsin, a red-sensitive cone visual pigment in the chicken retina.

The vertebrate retina contains two kinds of visual cells: rods, responsible for twilight (scotopic) vision (black and white discrimination); and cones, responsible for daylight (photopic) vision (color discrimination). Here we attempt to explain some of their functional differences and similarities in terms of their visual pigments. In the chicken retina there are four types of single cones and a double cone; each of the single cones has its own characteristic oil droplet (red, orange, blue, or colorless) and the double cone is composed of a set of principal and accessory members, the former of which has a green-colored oil droplet. Iodopsin, the chicken red-sensitive cone visual pigment, is located at outer segments of both the red single cones and the double cones, while the other single cones and the rod contain their own visual pigments with different absorption spectra. The diversity in absorption spectra among these visual pigments is caused by the difference in interaction between chromophore (11-cis retinal) and protein moiety (opsin). However, the chromophore-binding pocket in iodopsin is similar to that in rhodopsin. The difference in absorption maxima between both pigments could be explained by the difference in distances between the protonated Schiff-bases at the chromophore-binding site and their counter ions in iodopsin and rhodopsin. Furthermore, iodopsin has a unique chloride-binding site whose chloride ion serves for the red-shift of the absorption maximum of iodopsin. Visual pigment bleaches upon absorption of light through several intermediates and finally dissociates into all-trans retinal and opsin. That the sensitivity of cones is lower than rods cannot be explained by the relative photosensitivity of iodopsin to rhodopsin, but may be understood to some extent by the short lifetime of an enzymatically active intermediate (corresponding to metarhodopsin II) produced in the photobleaching process of iodopsin. The rapid formation and decay of the meta II-intermediate of iodopsin compared with metarhodopsin II are not contradictory to the rapid generation and recovery of cone receptor potential compared with rod receptor potential. The rapid recovery of the cone receptor potential may be due to a more effective shutoff mechanism of the visual excitation, including the phosphorylation of iodopsin. The rapid dark adaptation of cones compared with rods has been explained by the rapid regeneration of iodopsin from 11-cis retinal and opsin. One of the reasons for the rapid regeneration and susceptibility to chemicals of iodopsin compared with rhodopsin may be a unique structure near the chromophore-binding site of iodopsin.     

PHOTOCHEMISTRY OF METHYLATED RHODOPSINS

Abstract— Rhodopsin, in which the active-site Schiff-base lysine has been chemically modified by monomethylation, is unable to form the deprotonated Schiff base bleaching intermediate, rnetarhodop-sin II. The photochemistry of the methylated Schiff base rhodopsin stops at the metarhodopsin I stage, which then slowly decays to all-trans retinal and opsin. Methylation of the non active-site lysines does not block the photochemical transformation but does speed up the formation and decay of the metarhodopsins.