Effects of bleaching and regeneration on the purple membrane structure of Halobaterium halobium

Abstract

Sequential bleaching in the presence of hydroxylamine and subsequent regeneration of the purple membrane of Halobacterium halobium was studied by concomitant monitoring of its absorption and circular dichroic spectra in order to ascertain its effects on protein interaction(s) (which may result in possible excitonic interaction between the retinal chromophores), chromophore-apoprotein interaction(s), and protein conformational stability in the membrane. It was concluded that (a) although experimental results are consistent with an exciton mechanism for the interaction between retinal pi - pi* (NV(1)) transition movements in the purple membrane, no evidence for such a mechanism for interaction between retinaloxime transition moments is apparent in the case of the bleached membrane; (b) the bacteriorhodopsin molecules organized in clusters of three in the membrane appear to bleach simultaneously; (c) the retinaloxime produced on bleaching the purple membrane in the presence of hydroxylamine is strongly optically active, because of dissymmetry-inducing and/or -selecting constraints on the chromophore by a component of the membrane (most likely the apoprotein), and when the membrane is regenerated by the addition of retinal, these constraints are lost; and (d) evidence from ultraviolet absorption and circular dichroic spectra suggests that the membrane apoprotein undergoes appreciable conformational changes involving tertiary structure on bleaching with no significant secondary structure involvement. These results are compared with recently reported results from this laboratory on the effects of bleaching on the bovine rod outer segment disk membrane structure.