A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity

Abstract

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C₁₃-C₁₄ trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (λ_max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with λ_max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with λ_max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pK_a of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C₁₃-C₁₄ double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.