Pigment Analogs Research Articles

Relief of opsin desensitization and prolonged excitation of rod photoreceptors by 9-desmethylretinal.

The 9-methyl group of 11-cis-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretinal, combines with opsin to form a pigment that produces abnormal photoproducts and diminished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by approximately 7.5-fold to 0.15 +/- 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 +/- 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is approximately 30-fold smaller and decays approximately 5 times more slowly than that of native pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.

Azo pigments and their intermediates. A new class of couplers for red and near-IR sensitive photogenerating azo pigments

The synthesis of azoic couplers, 2,8-dihydroxy-3-naphthanilide and its derivatives, by first converting 2,8-dihydroxy-3-naphthoic acid into its phenyl ester followed by condensing the phenyl ester with an aniline derivative, is reported. Photogenerating bisazo and trisazo pigments have been prepared by coupling 2,8-dihydroxy-3-naphthanilide with 2,7-diamino fluorenone and tris( p-aminophenyl)amine, respectively. The absorption spectra of these pigments are found to be red-shifted relative to analogous pigments synthesized from 2-hydroxy-3-naphthanilide. The data suggest that 2,8-dihydroxy-3-naphthanilides are new coupler components for red and near-IR absorbing photogenerating azo pigments. Evidence is provided that the red shift is a result of an intramolecular H-bonding between the 8-OH group in the coupler moiety and the nitrogen atom in the hydrazone unit of the pigment. Comparison of the pigments synthesized in this work with those from 2-hydroxy-11 H-benzo(si)carbazole-3-carboxanilides suggests that the wavelength extension achieved by carbazole-couplers may also be a H-bonding effect, between the N-H group in the carbazole ring and the nitrogen in the hydrazone unit. The advantage of using the couplers described in this work for preparing red and near-IR absorbing photogenerating pigments is discussed.

Divergent pathways in photobleaching of 7,9-dicis-rhodopsin and 9,11-dicis-12-fluororhodopsin: One-photon-two-bond and one-photon-one-bond isomerization

Through low-temperature photochemistry, UV/vis spectroscopy, and chromophore extraction experiments, we have established that 7,9-dicis-rhodopsin undergoes one-photon-two-bond photoisomerization to a batho intermediate (its absorption maximum is slightly blue shifted from that of bathorhodopsin) containing the all-trans geometry, while 9,11-dicis-12-fluororhodopsin undergoes one-photon-one-bond isomerization to the corresponding 9-cis isomer and then the all-trans batho intermediate. The difference in the photochemical properties of the two dicis pigment analogs was rationalized by possible local protein perturbation, lability of the 11-cis geometry, and photochemical properties of the chromophores.

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

A NOVEL BACTERIORHODOPSIN ANALOGUE WITH CONFORMATIONALLY 6‐s‐cis FIXED RETINALS

Abstract— The artificial pigments of bacteriorhodopsin (bR) were synthesized with retinal analogues which have 6‐s‐cis fixed conformation. In order to study the shape of binding pocket around β‐ionone ring of retinal, we used three different 6‐s‐cis fixed retinals (1–3), which have a gem‐dimethyl group at a different position of the ring structure. These three 6‐s‐cis fixed retinals produced bR analogue pigments with absorption maxima at 574, 594 and 584 nm, respectively. These analogue pigments showed proton pumping activities upon incorporation into vesicles, indicating that 6‐s‐trans structure of retinal is not necessary to pump protons. Although these retinal analogues possess the same conjugated π‐electron systems, the wavelengths of maximum absorbance of the analogue pigments were different from each other, indicating a distinctly steric interaction between retinal ring portion and apoprotein. The analogue pigments were also different from each other in regeneration rate and the number of the final products. These differences resulted from a difference in the position of the dimethyl substituent at the ring structure. Based on the results the molecular structure around β‐ionone ring of retinal‐binding pocket was discussed.

Fluorine-19 NMR evidence for restricted rotation of the retinyl chromophore in doubly labeled visual pigment analogs

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTFluorine-19 NMR evidence for restricted rotation of the retinyl chromophore in doubly labeled visual pigment analogsLeticia U. Colmenares and Robert S. H. LiuCite this: J. Am. Chem. Soc. 1992, 114, 17, 6933–6934Publication Date (Print):August 1, 1992Publication History Published online1 May 2002Published inissue 1 August 1992https://doi.org/10.1021/ja00043a058RIGHTS & PERMISSIONSArticle Views41Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (286 KB) Get e-Alerts Get e-Alerts

EVIDENCE THAT THE REPELLENT RECEPTOR FORM OF SENSORY RHODOPSIN I IS AN ATTRACTANT SIGNALING STATE*

The lifetime of the Halobacterium halobium sensory rhodopsin I (SR-I) photocycle intermediate S373 was modulated by incorporating retinal analogs into SR-I apoprotein in vitro and in vivo. Photocycles by SR-I analog pigments exhibit the same reaction scheme and similar formation rates, but different decay rates, of their S373-like species as monitored by flash spectroscopy in membrane vesicle suspensions. The attractant receptor signaling efficiencies determined by physiological measurements are proportional to the lifetimes of the S373-like intermediates, indicating that S373 is a physiological active conformation (signaling state) of the receptor. A model incorporating this finding into the SR-I photocycle is presented.

RING OXIDIZED RETINALS FORM UNUSUAL BACTERIORHODOPSIN ANALOGUE PIGMENTS*

Three ring oxidized retinal analogues have been isolated from the exhaustive oxidation of all-trans retinal. All-trans 4-oxoretinal and 2,3-dehydro-4-oxoretinal have similar absorption maxima to that of all-trans retinal and have been shown to be in the 6-s-cis conformation in solution. Pigments formed with bacterioopsin exhibit absorption maxima (520 nm) blue-shifted from that of bacteriorhodopsin (bR), indicating a disturbance of the external point charge by the electronegative carbonyl moiety at the 4 position. The third analogue contains a ring contracted to a cyclopentenyl-alpha,beta-dione. Unlike the majority of retinals, this analogue displays a 6-s-trans conformation in solution and has a red-shifted absorption maximum at 435 nm. The resulting bR analogue pigment (515 nm) is formed five times faster than the other oxoretinal pigments. All three oxoretinal pigments show an irreversible 20 nm blue shift upon exposure to white light. The 4-oxo and 2,3-dehydro-4-oxoretinal pigments, after irradiation, undergo a small reversible blue shift (4-8 nm) on dark adaptation. These two pigments pump protons, although with slowed photocycle kinetics, demonstrating that these structural changes (addition of the carbonyl at the C-4 and insertion of a double bond in the ring) do not block the function of the pigment. Extraction of the C-15 tritiated analogue retinals from illuminated and non-illuminated pigments of all three oxoretinals yield identical results. Therefore, any crosslinking of these oxoretinals to the protein is by linkages which are unstable to the extraction procedures.

NMR studies of fluorinated visual pigment analogs

The 19F-nmr chemical shift data of isomeric pigments (11-cis and 9-cis) of four vinyl fluororhodopsins and two trifluororhodopsins have been recorded. When compared with model protonated Schiff bases, a set of F-nmr opsin shift parameter (FOS) was obtained. The data revealed regiospecific protein perturbations on the F-resonances. They can be interpreted in terms of specific protein interactions such as the postulated second point charge and other polar interactions as well as the common hydrophobic protein perturbation.

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogs

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)

11-Methyl-9-demethylretinal and 11-methyl-9,13-didemethylretinal. Effect of altered methyl substitution pattern on polyene conformation, photoisomerization and formation of visual pigment analogs

Altered methyl substitution pattern in 11-methyl-9-demethylretinal ( 1) and 11-methyl-9,13-didemethylretinal ( 2) resulted in changed polyene conformation, regioselectivity of photoisomerization, uv/vis characteristics and reactivity toward bovine opsin. That the 11-cis isomer of 1 and 9-cis isomer of 2 gave visual pigment analogs (λ max 475 and 459 nm respectively) while the 11-cis isomer of 2 did not is consistent with the requirement of a twisted chromophore near the middle of the polyene chain for pigment formation.

10,20‐Methanorhodopsins: (7E, 9E, 13E)‐10, 20‐methanorhodopsin and (7E, 9Z, 13Z)‐10, 20‐methanorhodopsin

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.

Effects of modifications of the retinal beta-ionone ring on archaebacterial sensory rhodopsin I

Ring desmethyl and acyclic analogues of all-trans retinal were incorporated into the apoprotein of the phototaxis receptor sensory rhodopsin I (SR-I) in Halobacterium halobium membranes. All modified retinals generate SR-I analogue pigments which exhibit "opsin shifts," i.e., their absorption spectra are shifted to longer wavelengths compared with model protonated Schiff bases of the same analogues. Each SR-I pigment analogue exhibits cyclic photochemical reactions as monitored by flash spectroscopy, but the analogue photocycles differ from that of native SR-I by exhibiting pronounced biphasic recovery of flash-induced absorption changes and abnormal flash-induced absorption difference spectra. Despite perturbations in the photochemical properties, the SR-I pigment analogues are capable of both attractant (single photon) and repellent (two photon) phototaxis signaling in cells. Our interpretation is that the hydrophobic ring substituents interact with the binding pocket to maintain the correct configuration for native SR-I absorption and photochemistry, but these interactions are not essential for the physiological function of SR-I as a dual attractant/repellent phototaxis receptor. These results support the conclusion emerging from several studies that the photoactivation process that triggers the conformation changes of SR-I and the related proton pump bacteriorhodopsin is conserved despite the different biological functions of their photoactivation.

Properties of apomembranes treated with sodium borohydride

The laser flash-induced photoelectric activity of apomembranes or pigment sheets and proteoliposomes associated with a phospholipid impregnated collodion film was measured. Two procedures were used to obtain apomembranes: (i) treatment of purple membranes with hydroxylamine (APOM. 1) and (ii) subsequent treatment of APOM. 1 with sodium borohydride in the dark (APOM. 2). It is shown that the treatment with sodium borohydride decreases the amount of residual bacteriorhodopsin significantly and that APOM. 2 can be used successfully for the regeneration of pigment analogues. However, experiments with one side inhibitor La 3+ ions demonstrate the presence of a heterogeneous orientation of sheets when the association of APOM. 2 with a planar film precedes pigment regeneration. It is concluded that regenerated pigments incorporated into proteoliposomes prior to association with a planar film are the most appropriate system for obtaining accurate measurements of the photoactivity of bacteriorhodopsin analogues.

Properties of apomembranes treated with sodium borohydride

The laser flash-induced photoelectric activity of apomembranes or pigment sheets and proteoliposomes associated with a phospholipid impregnated collodion film was measured. Two procedures were used to obtain apomembranes: (i) treatment of purple membranes with hydroxylamine (APOM. 1) and (ii) subsequent treatment of APOM. 1 with sodium borohydride in the dark (APOM. 2). It is shown that the treatment with sodium borohydride decreases the amount of residual bacteriorhodopsin significantly and that APOM. 2 can be used successfully for the regeneration of pigment analogues. However, experiments with one side inhibitor La 3+ ions demonstrate the presence of a heterogeneous orientation of sheets when the association of APOM. 2 with a planar film precedes pigment regeneration. It is concluded that regenerated pigments incorporated into proteoliposomes prior to association with a planar film are the most appropriate system for obtaining accurate measurements of the photoactivity of bacteriorhodopsin analogues.

Isomers, visual pigment, and bacteriorhodopsin analogs of 3,7,13-trimethyl-10-isopropyl-2,4,6,8,10-tetradecapentaenal and 3,7,11-trimethyl-10-isopropyl-2,4,6,8,10-dodecapentaenal (two ring open retinal analogs)

Six geometric isomers of 2 (3,7,13-trimethyl-10-isopropyl-2,4,6,8,10-tetradecapentaenal) have been isolated and characterized. Of these, four (equivalent to 9- cis, 11- cis, 5,9- dicis, and 5,11- dicis of retinoids) were found to form stable visual pigment analogs. So were four isomers (equivalent to 7- cis, 9- cis, 11- cis, and 9,13- dicis) of 3 (3,7,11-trimethyl-10-isopropyl-2,4,6,8,10-dodecapentaenal). These analogs suggest that the hydrophobic pocket is more flexible than what was recognized earlier, perhaps capable of accepting analogs with 6- S-trans as well as a 6- S-cis conformers. The presence of bacteriorhodopsin analogs from isomers of 2 was also reported.

Analog pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.

Halorhodopsin and sensory rhodopsin contain a C6-C7 s-trans retinal chromophore

Halorhodopsin (HR) and sensory rhodopsin (SR) have been regenerated with retinal analogues that are covalently locked in the 6-s-cis or 6-s-trans conformations. Both pigments regenerate more completely with the locked 6-s-trans retinal and produce analogue pigments with absorption maxima (577 nm for HR and 592 nm for SR) nearly identical to those of the native pigments (577 and 587 nm). This indicates that HR and SR bind retinal in the 6-s-trans conformation. The opsin shift for the locked 6-s-trans analogue in HR is 1,200 cm-1 less than that for the native chromophore (5,400 cm-1). The opsin shift for the 6-s-trans analogue in SR is 1,100 cm-1 less than that for the native retinal (5,700 cm-1). This demonstrates that approximately 20% of the opsin shift in these pigments arises from a protein-induced change in the chromophore conformation from twisted 6-s-cis in solution to planar 6-s-trans in the protein. The reduced opsin shift observed for the locked 6-s-cis analogue pigments compared with the locked 6-s-trans pigments may be due to a positive electrostatic perturbation near C7.

The shape of a three-dimensional binding site of rhodopsin based on molecular modeling analysis of isomeric and other visual pigment analogs. Bioorganic studies of visual pigments. 11

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe shape of a three-dimensional binding site of rhodopsin based on molecular modeling analysis of isomeric and other visual pigment analogs. Bioorganic studies of visual pigments. 11Robert S. H. Liu and Taraneh. MirzadeganCite this: J. Am. Chem. Soc. 1988, 110, 26, 8617–8623Publication Date (Print):December 1, 1988Publication History Published online1 May 2002Published inissue 1 December 1988https://doi.org/10.1021/ja00234a007RIGHTS & PERMISSIONSArticle Views134Altmetric-Citations45LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts