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

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Abstract
Synthesis of the retinal analog, 10,20‐methanoretinal (R6), where the 11Zconformation is locked in a six‐membered ring, yielded four stereoisomers (7E,9E,13E; 7E,9E,13Z, 7E,9Z,13Eand 7E,9Z,13Z). These four isomers were separated by straight‐phase isocratic HPLC and identified by1H‐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 13Eand 13Zisomers 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 (2660cm−1) is practically identical to that of rhodopsin itself (2650cm−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 > 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 11Zto all‐Eisomerization. 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 11‐cis‐locked rhodopsin analog pigments Rh6 and Iso6 are spectrally and structurally highly akin to rhodopsin, but lack its high photosensitivity and physiological activity. They could serve as suitable controls for rhodopsin in studies towards its light‐triggered functions or as suitable alternatives in studies on those properties which are not light‐dependent.

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