Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a sixmembered 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 'H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 1 1Z-retinal). The corresponding 13E and 1 3 2 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,92,13Z), i.e. equivalent to (9ZJ lZ,l3Z)-rhodopsin, an isorhodopsin analog. In analogy to the 1 I-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,92,132)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660cm-l) is practically identical to that of rhodopsin itself (2650 cm-'). Rh6 and Is06 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 1 1-cis-lock, Rh6 is much less photolabile (bleaching rate < 1 YO) 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 Is06 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 11 Z to all-E isomerization. Complete photostability and physiological inactivity could be achieved by substituting the Schiff-base link in Rh6 by an amide link, which is much less susceptible to hydrolytic cleavage, i.e. by recombining bovine opsin with (7E,9E, 13E)-10,20-methanoretinoyl fluoride.These results demonstrate that the six-membered ring 1 1-cis-locked rhodopsin analog pigments Rh6 and Is06 are spectrally and structurally highly akin to rhodopsin, but lack its high photosensitivity an...
ChemInform Abstract The retinal analogue (VI) with a fixed 11-cis configuration to preclude the light-induced 11-cis/trans isomerization and the retinoyl fluoride (VIII) are prepared by the procedures shown in the scheme (discussion of 1H, 13C, 19F NMR, MS and IR data). While (VIII) fails to react with bovine opsin, compound (VI) yields a light-and thermostable pigment (λmax= 390 nm) with the units being linked by a peptide bond rather than a Schiff base structure. The opsin shift of 2500 cm-1 is similar to that of rhodopsin (2650 cm-1), suggesting that the mechanism for the red shift is not located in the direct vicinity of the chromophore-protein linkage.
The binding of the isomers fall-trans, tfcis, f f-c& and 94s) of ~~~~~t~~lr~t~~~ to ~~~t~~i~o~~~~ and the light-dark adaptation as well as the tight-driven proton pump action of the resulting bacteriorhodopsin analogue were studied. The (Sdemethyl)-bacteriorhodopsin is formed -3-times faster than unmodified bacteriorhodopsin and shows an efficient light-driven proton pump action. These findings show that upon binding of retinal to bacterioopsin the protein forces the chromophore to adopt a more planar ring-chain conformation than in free retinal.~~obacterium hatobium &r@e mernb~~e Proton pumpChromoproteins with a retinylidene chromophore such as bacteriorhodapsin, visual pigments and retinochrome, play an important role in photobiology [I]. Bacteriorhodopsin (hereafter bR), the light energy converting protein of the purple membrane of the halo~hili~ microorganism Iiutobucreriwn halobium, occurs in a light-(Amax = 570 nm) and in a dark-adapted form (hmBX = 560 nm) [2]* The chromophore of the lightadapted form is an all-irons retinylidene moiety. In the dark-adapted form a 1: 1 ~~~ibrium between 13-t& and all-Pans isomers exists [Z].retinal does not bind to bO [Ltf* In continuation of our study on the effect of the different methy groups in retinal upon the binding properties and the light-driven proton pump action of bR [7], we now report on the binding properties of 5-demethyiretinai ( fig. 1) (in its dl-trans, 13-c&, 1 I-& and 9-c& form) to bO. The light-dark adaptation together with the light-driven proton pump action of (%demethyl)-bR are also discussed. 2, MATERIALS AND METHODS The
In order to obtain a light-stable rhodopsin as a potential candidate for crystallization and structural X-ray analysis, we synthesized and characterized the novel all E-10,20-methanoretinoyl fluoride (2). This retinal analogue has a locked 1 I-cis configuration, preventing the lightinduced 1 I-cis + trans isomerization and binds to opsin by a stable peptide bond rather than a Schiff base. 2 reacted with (methylated) bovine opsin, forming a light-and thermo-stable pigment with Alllax at 390 nm. Its opsin shift (2500 cm-' ) is in the same order of magnitude as that in rhodopsin (2650 cm-I), suggesting that the mechanism for the red shift is not located in the direct vicinity of the chromophore-protein linkage. We also report the first synthesis and characterization of all-E-retinoyl fluoride (lo), which failed to give a pigment on reaction with bovine opsin.
The synthesis of the title compound, a purple coloured retinoid having a fixed 11 -cis,l2s-cis-geometry, is described.t
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