Artificial Visual Pigment Research Articles

Structural Coupling of 11‐cis‐7‐Methyl‐retinal and Amino Acids at the Ligand Binding Pocket of Rhodopsin†

It was previously shown that opsin can be regenerated with the newly synthesized 11-cis-7-methyl-retinal forming an artificial visual pigment. We now extend this study to include mutants at positions close to the retinal to further dissect the interactions of native and artificial chromophores with opsin. Several mutants at M207, W265 and Y268 have been obtained and regenerated with 11-cis-retinal and the 7-methyl analog. M207 is the site of the point mutation M207R associated with the retinal degenerative disease retinitis pigmentosa. All the studied mutants regenerated with 11-cis-retinal except for M207C which proved to be completely misfolded. The naturally occurring M207R mutant formed a pigment with an unprotonated Schiff base linkage, altered photobleaching and low MetarhodopsinII stability. Mutants regenerated with the 7-methyl analog showed altered photobleaching reflecting a structural perturbation in the vicinity of M207. The newly obtained mutants at M207 also showed reduced levels of transducin activation with M207R showing essentially no transducin activation. Our results highlight the tight coupling of the vicinity of C7 of retinal and M207 and support the involvement of this amino acid residue in the conformational changes associated with rhodopsin photoactivation.

The Role of the 11‐cis‐Retinal Ring Methyl Substituents in Visual Pigment Formation

Artificial visual pigment formation from ring-demethylated retinals was studied in an effort to understand the effect that methyl groups on the chromophore cyclohexenyl ring have on the visual cycle. The stereoselective synthesis of the 11-cis-ring-demethylated analogues involves thallium-accelerated Suzuki cross-coupling reactions and highly stereocontrolled Wittig reactions to form key bonds. Only 11-cis-1,1,5-trisdemethylretinal (2) failed to form an artificial pigment, whilst variable pigment-formation yields were determined for the remaining analogues, increasing with the number (and location) of the chromophore hydrophobic ring methyl groups. Our results with the monodemethylated analogues 11-cis-5-demethylretinal (4) and 11-cis-1-demethylretinal (5) show that the C1-2-CH(3) groups are more important for pigment formation than the C5-CH(3) substituent. This is reflected in the absorption maxima of the artificial pigments, with values closer to that of native rhodopsin for 4. Docking studies based on a rhodopsin crystal structure, however, predict a lower pigment stability for 4 than for 5. Gas-phase DFT (B3LYP/6-31G*) computations of the free-ligand geometries, conformational searches about the C6--C7 bond, and docking studies revealed that, although the conformation of bound 5 is close to that of the native chromophore, the ligand needs to overcome the energy cost of shifting the unbound favored 6-s-trans conformation to the bound 6-s-cis form. In addition, the presence of an extra methyl group at C18 (11-cis-18-methylretinal, 7) is tolerated well and adds further stability to the complex, most probably due to increased hydrophobic interactions.

A methyl group at C7 of 11- cis-retinal allows chromophore formation but affects rhodopsin activation

The newly synthesized 11- cis-7-methylretinal can form an artificial visual pigment with kinetic and spectroscopic properties similar to the native pigment in the dark-state. However, its photobleaching behavior is altered, showing a Meta I-like photoproduct. This behavior reflects a steric constraint imposed by the 7-methyl group that affects the conformational change in the binding pocket as a result of retinal photoisomerization. Transducin activation is reduced, when compared to the native pigment with 11- cis-retinal. Molecular dynamics simulations suggest coupling of the C7 methyl group and the β-ionone ring with Met207 in transmembrane helix 5 in agreement with recent experimental results.

(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.

Two intermediates appear on the lumirhodopsin time scale after rhodopsin photoexcitation.

Absorbance difference spectra were recorded at 20 degrees C with a dense sequence of delay times from 1 to 128 micros after photolysis of lauryl maltoside suspensions of rhodopsin prepared from hypotonically washed bovine rod outer segments. Data were best fit by two-exponential components with a small, fast component (tau = 12 micros) occurring during the period that lumirhodopsin has been presumed to be stable. The shape of the spectral change corresponds to an approximately 2 nm red shift of the lumirhodopsin spectrum. Measurements with linearly polarized light verified that no absorbance changes associated with rotational diffusion were present in these preparations on this time scale, and experiments designed to enhance isorhodopsin production during photolysis showed no effect on the relative amplitude of the fast process. A similar process was previously observed in membrane suspensions of rhodopsin, but there the similarity of the change to rotational diffusion artifacts made conclusive identification of a second lumirhodopsin difficult. However, reexamination of polarized light measurements on rhodopsin in membrane supports the fact that the fast process seen here in detergent also takes place there. The new absorbance process occurs when time-resolved resonance Raman experiments have shown that the protonated Schiff base is moving from one hydrogen bond acceptor to another. The results are discussed in the context of possibly related processes on the same time scale that have been observed recently in artificial visual pigments with synthetic retinylidene chromophores and in a related rhodopsin mutant. The details of lumirhodopsin behavior are important because it is the last protonated Schiff base intermediate that occurs under physiological conditions.

A Nonbleachable Rhodopsin Analogue with a Slow Photocycle

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.

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection.

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.

Steric barrier to bathorhodopsin decay in 5-demethyl and mesityl analogues of rhodopsin.

Absorbance difference spectra were recorded from 20 ns to 1 micros after 20 degrees C photoexcitation of artificial visual pigments derived either from 5-demethylretinal or from a mesityl analogue of retinal. Both pigments produced an early photointermediate similar to bovine bathorhodopsin (Batho). In both cases the Batho analogue decayed to a lumirhodopsin (Lumi) analogue via a blue-shifted intermediate, BSI, which formed an equilibrium with the Batho analogue. The stability of 5-demethyl Batho, even though the C8-hydrogen of the polyene chain cannot interact with a ring C5-methyl group to provide a barrier to Batho decay, raises the possibility that the 5-demethylretinal ring binds oppositely from normal to form a pigment with a 6-s-trans ring-chain conformation. If 6-s-trans binding occurred, the ring C1-methyls could replace the C5-methyl in its interaction with the chain C8-hydrogen to preserve the steric barrier to Batho decay, consistent with the kinetic results. The possibility of 6-s-trans binding for 5-demethylretinal also could account for the unexpected blue shift of 5-demethyl visual pigments and could explain why 5-demethyl artificial pigments regenerate so slowly. Although the mesityl analogue BSI's absorption spectrum was blue-shifted relative to its pigment spectrum, the blue shift was much smaller than for rhodopsin's or 5-demethylisorhodopsin's BSI. This suggests that increased C6-C7 torsion may be responsible for some of BSI's blue shift, which is not the case for mesityl analogue BSI either because of reduced spectral sensitivity to C6-C7 torsion or because the symmetry of the mesityl retinal analogue precludes having 6-s-cis and 6-s-trans conformers. The similarity of the mesityl analogue BSI and native BSI lambda(max) values supports the idea that BSI has a 6-s angle near 90 degrees, a condition which could disconnect the chain (and BSI's spectrum) from the double bond specifics of the ring.

Salt Dependence of the Formation and Stability of the Signaling State in G Protein-Coupled Receptors: Evidence for the Involvement of the Hofmeister Effect

We studied the salt dependence of both the stability and the equilibrium of the late photoproducts metarhodopsin I (MI) and II (MII) of the artificial visual pigment 9-demethyl rhodopsin (9dm-Rho). In the photoproducts of 9dm-Rho, all-trans-9-demethyl retinal acts only as a partial agonist, enabling us to study the photoproduct equilibrium of the pigment both in membranes and in detergent micelles. Chloride, bromide, and phosphate salts shift this equilibrium from the inactive MI to the active MII receptor conformation both in native membranes and even more with purified pigment in detergent micelles. In the presence of these salts, the induced MII state seems to be structurally intact, as judged by Fourier transform infrared (FTIR) and UV-vis spectroscopy. In the long term, however, we observe an increased instability of the photoproducts and a change in the decay pathways. Both MII enhancement and destabilization are particularly pronounced with the strong chaotropic salts KI and KSCN. The results fit into the framework of the Hofmeister effect and are assigned to an increased solvation of the peptide moiety of the solvent-exposed domains, their resulting partial disordering favoring MII over MI. In this picture, increased solvation also affects helix-helix interactions, thereby leading to a structural instability of the protein in the long term. The reported influences of salts on conformation and stability of this membrane protein are likely to be general and may therefore also apply to other transmembrane proteins and particularly to other G protein-coupled receptors.

Early photolysis intermediates of gecko and bovine artificial visual pigments.

Nanosecond laser photolysis measurements were conducted on digitonin extracts of artificial pigments prepared from the cone-type visual pigment, P521, of the Tokay gecko (Gekko gekko) retina. Artificial pigments were prepared by regeneration of bleached gecko photoreceptor membranes with 9-cis-retinal, 9-cis-14-methylretinal, or 9-cis-alpha-retinal. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 microseconds following excitation with a pulse of 477-nm actinic light. Global analysis showed the kinetic data for all three artificial gecko pigments to be best fit by two-exponential processes. These two-exponential decays correspond to similar decays observed after photolysis of P521 itself, with the first process being the decay of the equilibrated P521 Batho<-->P521 BSI mixture to P521 Lumi and the second process being the decay of P521 Lumi to P521 Meta I. In spite of its large blue shift relative to P521, iso-P521 displays a normal chloride depletion induced blue shift. Iso-P521's early intermediates up to Lumi were also blue-shifted, with the P521 Batho<-->P521 BSI equilibrated mixture being 15 nm blue-shifted and P521 Lumi being 8 nm blue-shifted relative to the intermediates formed after P521 photolysis. The blue shift associated with the iso-pigment is reduced or disappears entirely by P521 Meta I. Similar blue shifts were observed for the early intermediates observed after photolysis of bovine isorhodopsin, with the Lumi intermediate blue-shifted 5 nm compared to the Lumi intermediate formed after photolysis of bovine rhodopsin. These shifts indicate that a difference exists between the binding sites of 9- and 11-cis pigments which persists for microseconds at 20 degrees C.

Physiological Activity of Retinoids in Natural and Artificial Visual Pigments

Photochemistry and PhotobiologyVolume 63, Issue 5 p. 595-600 Free Access Physiological Activity of Retinoids in Natural and Artificial Visual Pigments D. Wesley Corson, Corresponding Author D. Wesley Corson Department of Pathology and Laboratory Medicine Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA*Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA. Fax: 803–792–1723; e-mail: CORSONDW@MUSC.EDU.Search for more papers by this authorRosalie K. Crouch, Rosalie K. Crouch Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USASearch for more papers by this author D. Wesley Corson, Corresponding Author D. Wesley Corson Department of Pathology and Laboratory Medicine Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA*Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA. Fax: 803–792–1723; e-mail: CORSONDW@MUSC.EDU.Search for more papers by this authorRosalie K. Crouch, Rosalie K. Crouch Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USASearch for more papers by this author First published: May 1996 https://doi.org/10.1111/j.1751-1097.1996.tb05661.xCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume63, Issue5May 1996Pages 595-600 ReferencesRelatedInformation

Low-temperature trapping of early photointermediates of alpha-isorhodopsin.

Alpha-Isorhodopsin, an artificial visual pigment with a 9-cis-4,5-dehydro-5,6-dihydro(alpha)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K,a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rhodopsin, Batho is the first intermediate detected in alpha-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. Alpha-Isohodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C5-C6 double bond. The transition temperature for BSI formation is higher in alpha-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.

ChemInform Abstract: A New Photolysis Intermediate in Artificial and Native Visual Pigments.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

FTIR evidence of an altered chromophore-protein interaction in the artificial visual pigment cis-5,6-dihydroisorhodopsin and its photoproducts BSI, lumirhodopsin, and metarhodopsin-I

FTIR evidence of an altered chromophore-protein interaction in the artificial visual pigment cis-5,6-dihydroisorhodopsin and its photoproducts BSI, lumirhodopsin, and metarhodopsin-I

Early photolysis intermediates of the artificial visual pigment 13-demethylrhodopsin

Nanosecond time-resolved absorption measurements are reported for the room temperature photolysis of a modified rhodopsin pigment, 13-demethylrhodopsin, which contains the chromophore 13-demethylretinal. The measurements are consistent with the formation of an equilibrium between a BA-THO-13-demethylrhodopsin species and a blue-shifted species (relative to the parent pigment), BSI-13-demethylrhodopsin. The results are compared to those acquired after photolysis of native bovine rhodopsin [Hug, S. J., Lewis, J. W., Einterz, C. M., Thorgeirsson, T. E., & Kliger, D. S. (1990) Biochemistry (preceding paper in this issue)] and to results obtained after photolysis of several modified isorhodopsin pigments in which the BSI species was first observed. It is concluded that in all of the pigments the results are consistent with the formation of an equilibrium between BATHO and BSI, which subsequently decays on a nanosecond time scale at room temperature to a lumirhodopsin intermediate.

Nanosecond photolysis of rhodopsin: evidence for a new blue-shifted intermediate

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.

Photolysis intermediates of the artificial visual pigment cis-5,6-dihydro-isorhodopsin

The photolysis intermediates of an artificial bovine rhodopsin pigment, cis-5,6-dihydro-isorhodopsin (cis-5,6,-diH-ISORHO, lambda max 461 nm), which contains a cis-5,6-dihydro-9-cis-retinal chromophore, are investigated by room temperature, nanosecond laser photolysis, and low temperature irradiation studies. The observations are discussed both in terms of low temperature experiments of Yoshizawa and co-workers on trans-5,6-diH-ISORHO (Yoshizawa, T., Y. Shichida, and S. Matuoka. 1984. Vision Res. 24: 1455-1463), and in relation to the photolysis intermediates of native bovine rhodopsin (RHO). It is suggested that in 5,6-diH-ISORHO, a primary bathorhodopsin intermediate analogous to the bathorhodopsin intermediate (BATHO) of the native pigment, rapidly converts to a blue-shifted intermediate (BSI, lambda max 430 nm) which is not observed after photolysis of native rhodopsin. The analogs from lumirhodopsin (LUMI) to meta-II rhodopsin (META-II) are generated subsequent to BSI, similar to their generation from BATHO in the native pigment. It is proposed that the retinal chromophore in the bathorhodopsin stage of 5,6-diH-ISORHO is relieved of strain induced by the primary cis to trans isomerization by undergoing a geometrical rearrangement of the retinal. Such a rearrangement, which leads to BSI, would not take place so rapidly in the native pigment due to ring-protein interactions. In the native pigment, the strain in BATHO would be relieved only on a longer time scale, via a process with a rate determined by protein relaxation.

ChemInform Abstract: An Artificial Visual Pigment with Restricted C9‐C11 Motion Forms Normal Photolysis Intermediates.

AbstractThe artificial 11‐cis‐retinal (I) (synthesis already published) forms a pigment when incubated with bovine opsin.

An artificial visual pigment with restricted carbon-9-carbon-11 motion forms normal photolysis intermediates

An artificial visual pigment with restricted carbon-9-carbon-11 motion forms normal photolysis intermediates

A microspectrophotometric study of normal and artificial visual pigments in the photoreceptors of Xenopus laevis

Absorbance spectra of visual pigments in the Xenopus retina were obtained by microspectrophotometry. Principal rods contained a pigment with peak absorbance of 524 ± 2nm. A minority of rods contained a pigment with λ max at 445 ± 6nm; single cone spectra peaked at 611 ± 4nm. Xenopus tadpoles were maintained on a Vitamin A-free diet for 6–8 weeks, then injected systemically with all-trans retinol or 9-cis retinal. In the principal rod, these resulted in pigments peaking at 511 ± 3nm and 498 ± 2nm, respectively. The normally encountered Vitamin A 2-based pigment of the principal rod had a broader half-width than its Vitamin A 1-based analog. In single cones, all-trans retinol led to the formation of a pigment with λ max 578 ± 7nm. The substantial shifts seen in the wavelengths of peak absorbance as well as in the shapes of the spectral curves following administration of retinol or 9-cis retinal indicate that at least most of the normally encountered Vitamin A 2-based pigment was replaced by a Vitamin A 1-based pigment.