Cattle Opsin Research Articles

Differences in the photobleaching process between 7-cis- and 11-cis-rhodopsins: a unique interaction change between the chromophore and the protein during the lumi-meta I transition

The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed.

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

Incubation of purified 9,13-dicis-retinal with cattle opsin in 2% digitonin at 20 degrees C produced two pigments, one unstable (lambda max 478 nm) and the other stable (lambda max 485 nm) in hydroxylamine. The two pigments exhibited different characteristics. HPLC analysis revealed that the chromophores of these pigments have respectively 9,13-dicis and 9-cis geometries. Under various conditions the amount of 9,13-dicis-rhodopsin formed never exceeded 30% of the total pigments. The addition of 9,13-dicis-retinal to the ROS suspension containing opsin produced 9-cis-rhodopsin in 97% yield. Irradiation of the 9,13-dicis-rhodopsin mainly produced 13-cis-retinal, while 9-cis-rhodopsin produced the all-trans isomer. These results demonstrated that the one-photon-one-double-bond isomerization process took place in 9,13-dicis-rhodopsin.

Electrostatic interaction between retinylidene chromophore and opsin in rhodopsin studied by fluorinated rhodopsin analogues.

Photochemical reactions of fluorinated rhodopsin analogues (F-rhodopsins) prepared from 10- or 12-fluorinated retinals (10- or 12-F-retinals) and cattle opsin were investigated by means of low-temperature spectrophotometry. On irradiation with blue light at liquid nitrogen temperature (-191 degrees C), the F-rhodopsins were converted to their respective batho intermediates. On warming, they decomposed to their respective fluororetinals and cattle opsin through lumi and meta intermediates. There was a difference in photochemical behavior between batho-12-F-rhodopsin and batho-10-F-rhodopsin. Upon irradiation with red light at -191 degrees C, batho-12-F-rhodopsin was converted to a mixture of 12-F-rhodopsin and 9-cis-12-F-rhodopsin like that of the natural bathorhodopsin, whereas batho-10-F-rhodopsin was not converted to 9-cis-10-F-rhodopsin but only to 10-F-rhodopsin. This fact suggests that the fluorine substituent at the C10 position (i.e., 10-fluoro) of the retinylidene chromophore may interact with the protein moiety during the process of isomerization of the chromophore or in the state of the batho intermediate. On irradiation with blue light at -191 degrees C, 9-cis-10-F-rhodopsin was converted to another bathochromic intermediate that was different in absorption spectrum from batho-10-F-rhodopsin. 9-cis-10-F-rhodopsin was practically "photoinsensitive" at liquid helium temperature (-265 degrees C), whereas 10-F-rhodopsin was converted to a photo-steady-state mixture of 10-F-rhodopsin and batho-10-F-rhodopsin. The specific interaction between the fluorine atom at the C10 position of the retinylidene chromophore and the opsin was discussed in terms of electrostatic interactions.

Low temperature spectrophotometric study of cattle bathorhodopsins produced from rhodopsin and isorhodopsin in transparent medium without cracks.

Abstract— Cattle rhodopsin (11‐cis‐Rh) and isorhodopsin (9‐cis‐Rh) were prepared from the same extract of cattle opsin by incubation in the dark with 114– and 9‐cis‐retinals, respectively. The preparations obtained were mixed with glycerol (67% in the final concentration) and then cooled to 77 K by a rapid cooling method for freezing the samples as a clear glass, without any cracks. The spectral changes during their photochemical reactions were measured. A computer analysis of the spectra obtained verified that bathorhodopsin from 11‐cis‐Rh was identical in spectrum with that from 9‐cis‐Rh not only in photosteady state but also under a short time irradiation.

Chiral discrimination of 5,6-epoxy-3-dehydroretinal by aporetinochrome and cattle opsin.

The combination of a racemic all-trans 5,6-epoxy-3-dehydroretinal (EDR) with aporetinochrome formed a mixture of two diastereomeric pigments. One of the diastereomeric pigments which contained the all-trans EDR with a negative circular dichroic (CD) band, hereafter called EDR(-)-chrome, has its visible absorption maximum around 438 nm, while the other pigment, called EDR(+)-chrome, has its maximum at 464 nm. These were substantiated by measuring the optical activities of the two EDR isomers which were extracted from a mixture of racemic all-trans EDR and a smaller amount of aporetinochrome following exposure to orange light (greater than 530 nm) that irradiates EDR(+)-chrome selectively. The extracted all-trans EDR had a negative CD band around 240 nm and the extracted 11-cis EDR had a positive band in that region with two negative bands on either side of the main band. In the case of both pigments, the effect of alkalinization on the increase of absorbance in the near-ultraviolet and the decrease of absorbance in the visible region was proportionate, qualitatively, to that on the positive CD intensities in both regions. These results suggest that the chromophore EDR in each pigment binds to the same binding site via a Schiff base. The EDR(+)-chrome exhibited properties similar to those of retinochrome, but EDR(-)-chrome showed some different properties, i.e. its formation rate was slower than that of the former one and its absorption band in the near-ultraviolet appeared even at neutral pH. Moreover, by exposing EDR(-)-chrome to yellow light (greater than 480 nm), only a part of its prosthetic all-trans EDR was isomerized and resulted in the formation of 11-cis and 13-cis isomers. This variation in photoisomerizing activity was supposed to be due to the difference in conformation of the side chain between EDR(+) and EDR(-) in aporetinochrome. Combination of 11-cis EDR with cattle opsin was also shown to result in the formation of two diastereomeric pigments. The absorption maxima of the diastereomers containing 11-cis EDR(+) and EDR(-) were at about 446 and 474 nm, respectively.

Competition between retinal and 3-dehydroretinal for opsin in the regeneration of visual pigment

Rhodopsin regenerated faster than porphyropsin in all preparations of bullfrog opsin. bullfrog rod outer segment membrane and cattle opsin. When opsin was incubated with excess amount of an equimolar mixture of 11- cis-retinal and 11- cis-3-dehydroretinal, the composition of the regenerated pigment was simply dependent on the ratio of regeneration rates of rhodopsin and porphyropsin. This result can provide a mechanism to account for the discrepancy in vitamin A 1/ A 2 composition between the retina and the pigment epithelium. The property of opsin preferring retinal to 3-dehydroretinal may be one of the basic factors affecting vitamin A 1/A 2 visual pigment systems.

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin.

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.

Photophysiological functions of visual pigments.

In order to gain a fundamental understanding of functions of a visual pigment, i.e., photoreception and phototransduction, it is essential to elucidate the molecular structure of visual pigment, its photochemical behavior and connection of the pigment to the molecular physiological amplification mechanism for excitation of a visual cell. A rhodopsin, a rod visual pigment, is composed of an 11-cis-retinal bound with an apo-protein, opsin, through a protonated Schiff-base. Competitive inhibition of beta-ionone on regeneration of rhodopsin from an 11-cis-retinal and cattle opsin demonstrated the existence of a hydrophobic linkage between the beta-ionone ring of the retinal and the hydrophobic region of opsin. Owing to these two linkages, the 11-cis-retinal is fixed in an opsin cleft. As a result, it is endowed with new physiological functions as a chromophore of rhodopsin; the change of 11-cis-retinal (lambda max = 369 nm, epsilon = 26,400) to rhodopsin (lambda max = about 500 nm, epsilon = 40,600) brings not only a spectral shift from near ultraviolet to visible regions and an intensification of the molecular extinction coefficient, but also an increase of a quantum yield of 11-cis-retinal to all-trans form. High quantum yield of rhodopsin suggests rapid formation of the first photoproduct. Study of the first photoproduct was accelerated by the finding of bathorhodopsin, which was formed by irradiation at liquid nitrogen temperatures. The change of rhodopsin to bathorhodopsin has been inferred to be due to a photoisomerization of the chromophore from 11-cis to a twisted all-trans form. This isomerization hypothesis has been verified by the following experimental results. Irradiation of 7-cis- and 9-cis-rhodopsins at liquid nitrogen temperature produced the same bathorhodopsin as that from 11-cis-rhodopsin, indicating that the chromophore of bathorhodopsin should be in all-trans or transoid form. 7-Membered-rhodopsin, in which the rotation of 11-12 double bond of the retinylidene chromophore is locked, did not form bathorhodopsin by excitation of picosecond laser photolysis. This fact indicates that bathorhodopsin is a product formed by photoisomerization of the chromophore. Rhodopsin showed a positive circular dichroism (CD) in the visible while bathorhodopsin showed a remarkable negative CD. The reversal of the sign of CD indicates that not only large conformational change of the retinylidene chromophore occurs during the conversion of rhodopsin to bathorhodopsin, but also the direction of twist of the chromophore reverses. It is inferred that the chromophore of bathorhodopsin is a twisted trans form.(ABSTRACT TRUNCATED AT 400 WORDS)

Visual pigment analogs from isomers of 5,6,7,8-tetrahydroretinal. The importance of the trimethylcyclohexyl ring

Three of the five isomers of 5,6,7,8-tetrahydroretinal were found to form pigment analogs when incubated with cattle opsin. Failure of dehydrocitral to form a similar pigment indicates the importance of the six-membered ring in pigment formation.

Reconstitution of squid and cattle rhodospin by the use of metaretinochrome in their respective membranes

Retinochrome is, even in membranes, converted to metaretinochrome by exposure to orange light. Upon incubation of metaretinochrome in membranes with cattle opsin in rod outer segment membranes, cattle rhodopsin is reconstituted in the dark. When opsin is present in molar excess to metaretinochrome, about 80% of the prosthetic retinal of retinochrome present initially is utilized for the reconstitution of cattle rhodopsin. One reason why all of the prosthetic retinal is not used for the rhodopsin reconstitution is that metaretinochrome transforms slowly to retinochrome during incubation in the dark and another is that metaretinochrome is in a photoequilibrium mixture with a trace of retinochrome after exposure to orange light. Squid rhodopsin is reconstituted when a mixture of metaretinochrome and squid opsin in their respective membranes is incubated in the dark. The reconstituted rhodopsin is converted to acid or alkaline metarhodopsin by exposure to orange light at neutral or alkaline pH, respectively. Three possible mechanisms for the transference of 11- cis retinal from metaretinochrome in a membrane to opsin in a different membrane were considered: (1) the migration of 11- cis retinal through an aqueous medium between the separate membranes, (2) the migration of 11- cis retinal from metaretinochrome to opsin in a fused membrane and (3) the transfer of retinal from membrane to membrane in close contact. In conclusion, the first two mechanisms were inapplicable and the third appeared to explain the present experimental findings. The possibility is discussed that the photoproduct of retinochrome may contribute to the rhodopsin synthesis as an effective donor of 11- cis retinal to opsin in the squid retina.

93] Fluorine-labeled retinals and rhodopsins

Publisher Summary This chapter discusses the fluorine (F)-labeled retinals and rhodopsins. The use of F as a label to probe for structural information of complex biomolecules by nuclear magnetic resonance (nmr) spectroscopy is an established technique. Its advantages over other NMR active nuclei are several. Among them is the absence of background signals from proteins or detergents. The disadvantages associated with the method are the synthetic difficulties sometimes encountered in introducing the fluorine labels at strategic positions and the possibility that the highly electronegative fluorine atom might change the overall properties of the biomolecules of interest. The absorption maxima of isomers of several fluorinated rhodopsins are listed in the accompanying table along with those of the corresponding retinals. That the selectivity of fluororetinal isomers in their reaction with cattle opsin is virtually the same as the parent retinals suggests that the fluorine substituents do not exert a vastly different steric effect.

Photochemical reactions of 13-demethyl visual pigment analogues at low temperatures.

The photobleaching reaction of 13-demethylisorhodopsin (hereafter designated as 9-cis- 13-dm-rhodopsin), which was synthesized from 9-cis- 13-demethylretinal and cattle opsin, was investigated by low-temperature spectrophotometry in order to elucidate the role of the 13-methyl group of retinal in photobleaching. When 9-cis- 13-dm-rhodopsin was irradiated at-190 degrees C, batho-13-dm-rhodopsin was produced. Its absorption maximum lay at 532 nm, 11 nm shorter than that of cattle bathorhodopsin (gamma max 543 nm), and batho-13-dm-rhodopsin had an extinction coefficient about 0.6 times that of bathorhodopsin. Batho-13-dm-rhodopsin was thermally unstable. Above-180 degrees C, it converted to a new intermediate, BL-13-dm-rhodopsin, which in turn changed to lumi-13-dm-rhodopsin- above -140 degrees C. BL-13-dm-rhodopsin was "photosensitive" at temperatures around -188 degrees C, though batho-13-dm-rhodopsin and lumi-13-dm-rhodopsin was "photosensitive" at the same temperature. In the photobleaching process, lumi-13-dm-rhodopsin and meta-I-13-dm-rhodopsin were observed. Their thermostabilities were very similar to those of lumirhodopsin and metarhodopsin I, but each dm intermediate differed from its methylated counterpart in its value of gamma max and extinction coefficient.

Aromatic retinal analogues and their interaction with cattle opsin.

The preparation of seven aromatic analogues of retinal, the isolation and characterization of their geometric isomers, and the interaction of these isomers with cattle opsin are reported. Within certain limitations, it has been demonstrated that stable aromatic rhodopsin analogues can be prepared. In general, the stereoselectivity of isomers of these compounds in their interaction with opsin and the absorption properties of the resultant pigments are similar to those of the parent retinal.

Photochemical studies of 7-cis-rhodopsin at low temperatures. Nature and properties of the bathointermediate.

The photoreaction of 7-cis-rhodopsin derived from 7-cis-retinal and cattle opsin was studied by low-temperature spectrophotometry. Upon irradiation of 7-cis-rhodopsin at liquid nitrogen temperature (-190 degrees C) with blue light, its spectrum shifted to the longer wavelengths, indicating the formation of a bathoproduct. The bathoproduct thus formed was found to be identical with bathorhodopsin formed from rhodopsin in their spectroscopic, photochemical, and thermal properties. Therefore, we believe that the bathoproduct is, in fact, bathorhodopsin. The fact that 7-cis-rhodopsin can be readily converted to rhodopsin and to 9-cis-rhodopsin shows that the identical retinal binding site of opsin is involved in the three isomeric rhodopsins. These results appear to be consistent with the notion that the chromophore of bathorhodopsin is a twisted all-trans isomer, which is readily obtainable from the 7-cis, 9-cis, and 11-cis isomers.

Photochemical reaction of 9- cis-retro-γ-rhodopsin at low temperatures

9- cis-Retro-γ;rhodopsin ( λ max = 420 nm) was prepared from 9- cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11- cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.

Doubly hindered 7,11-dicis isomers of retinal. Synthesis, properties, and interaction with cattle opsin

Doubly hindered 7,11-dicis isomers of retinal. Synthesis, properties, and interaction with cattle opsin

Formation of 7-cis- and 13-cis-retinal pigments by irradiating squid rhodopsin

Squid rhodopsin was irradiated with orange light (greater than 530 nm) at various temperatures from -190 to 10 degrees C until a photo-steady-state mixture was formed. Then the chromophoric retinals were extracted from the photo-steady-state mixtures and their isomer composition was analyzed by high-performance liquid chromatography. In the case of photo-steady-state mixture formed at -85 degrees C, large peaks in the chromatogram were found at the positions of both 7-cis- and 13-cis-retinals. Each peak was further identified by synthesizing the pigments from these retinals with cattle opsin or apobacteriorhodopsin. Both 7-cis- and 13-cis-retinals were also extracted from a photo-steady-state mixture formed by irradiation at -40, at 0, or at 10 degrees C. These isomers were scarcely detected in a photo-steady-state mixture formed by irradiation at -190 degrees C, though 9-cis-retinal was found as a major constituent in this mixture. Irradiation of lumirhodopsin at -190 degrees C, however, produced 7-cis-retinal pigment. These findings suggest that bathorhodopsin may have a conformation to prevent the formation of 7-cis-retinal from the all-trans form and that this particular conformation may be relaxed by the conversion of bathorhodopsin to lumirhodopsin.

7‐cis‐PORPHYROPSIN FROM 7‐cis‐3‐DEHYDRORETINAL AND CATTLE OPSIN*

Abstract 7‐cis‐3‐Dehydroretinol, prepared by photoisomerization of the all‐trans isomer in a polar solvent, was found to combine with cattle opsin to form a visual pigment analogue with an absorption maximum at 464 nm. The pigment is only moderately stable in hydroxylamine or in Ammonyx LO. and cannot be purified by column chromatography using Ammonyx LO. 9‐cis‐Porphyropsin, prepared in a manner similar to that reported by Azuma et al., is stable and has been purified by passing through a hydroxylapatite column. Three fractions were collected with eluant of increasing ionic strength. All fractions exhibit similar absorption properties with Λmax at 498 nm. These results further indicate that the binding of opsin is a flexible one.

Properties of Opsin-Lipid Complex in Aqueous 2-Chloroethanol

Cattle and squid opsins were found to be associated with phospholipids after extensive dialysis of the salt-free digitonin extract of rhodopsin against 30% aqueous 2-chloroethanol (v/v) at pH 2.5. The approximates sizes of opsin-lipid complexes were estimated by sedimentation studies to be around 110,000 and 150,000 daltons, respectively, for the cattle and the squid opsin. Phospholipids did not dissociate from opsin even in 80% 2-chloroethanol. The complexes were purified by passage through a Sephadex G-200 gel column equilibrated with 30% 2-chloroethanol. The optical properties of the complex suggested the presence of beta-conformation and a small amount of alpha-helix in solubilized cattle opsin.

Rhodopsin analogues from highly hindered 7-cis isomers of retinal.

ALL known visual pigments have as chromophore the sterically hindered 11-cis isomer of retinal. Shortly after this was established, an unsuccessful attempt was made to prepare the much more hindered 7-cis-retinal by partial reduction of the corresponding 7,8-acetylene1. But, four new geometric isomers of retinal, all apparently 7-cis, have been synthesised by a six-step non-stereospecific sequence2. Structural assignments of the isomers were originally based on spectral data of two fractions of partially separated mixtures of isomers (7-cis and 7,9-dicis; 7,13-dicis and 7,9,13-tricis). Their configurations were, therefore, not completely firm. Since then two of the four isomers (7-cis and 7,9-dicis) have been isolated by high pressure liquid chromatography (HPLC). Their nuclear magnetic resonance (NMR) spectra unambiguously confirmed the tentative assignments made earlier. That of 7-cis-retinal is shown in Fig. 1 as a representative example. The remaining two 13-cis isomers remain unseparated in various HPLC conditions. Their structures, however, have since been confirmed through a steroselective synthesis of 7,9,13-tricis-retinal3. We report here preliminary results of the interaction of these 7-cis-retinal isomers with cattle opsin.