Sensory rhodopsins in Halobacterium halobium: Studies in vivo and in vitro

Abstract

Halobacterium halobium is a halophilic archaebacterium which has been intensively studied in recent years for a family of retinal-containing membrane proteins which are light-receptors for the primary energetic needs of the cell and for its photosensitive swimming behavior. Bacteriorhodopsin (bR) and halorhodopsin (hR), are light-driven ion pumps, while sensory rhodopsin I and II serve as photoreceptors mediating light-induced motility responses exhibited by the cells.;The work is presented in four chapters:;Chapter I. Preparations for the study of halobacterial rhodopsins. When these studies began, the first of the sensory rhodopsins (sR-I) had been detected only in isolated membranes. This chapter describes a comparative study of preparations which maintain sR-I activity: intact cells, sonicated cell envelopes, and membrane fraction prepared by dialysis. These materials were tested for the amount of pigment present and photochemical and biochemical properties. The advantages and shortcomings of each preparation for specific experimental purposes undertaken in later chapters are discussed.;Chapter II. Photochemical properties of sR-I in sonicated vesicles and in detergent. The photochemical reactions of sR-I are described in sonicated cell envelopes and in detergents. The effect of external pH on the absorption and photocycle of sR-I are described, revealing a titratable basic group (pK{dollar}\sb{lcub}\rm app{rcub}{dollar} = {dollar}\sim{dollar}8.5) that interacts with the retinal chromophore and affects its absorption maximum and its photocycle kinetics.;Chapter III. Effects of membrane potential on the photocycling rates of three halobacterial retinal proteins. In the course of these investigations, substantial differences between the sR-I photoreaction kinetics in vivo from those in membranes were found. We determined that the difference could be attributed to the electrical potential across the cell membrane in vivo. In vivo sR-I photocycling rates were reproduced in envelope vesicle preparations in the presence of valinomycin-induced potassium diffusion potentials. Comparative analysis of bR, hR, and sR-I revealed each of the three pigments exhibits a decreased rate of thermal decay of its principal intermediate when photoactivated in an artifically energized compared to de-energized membrane, suggesting a voltage-dependent conformational change common to their photocycles.;Chapter IV. Enrichment of sR-I. In this part different strategies are described for the enrichment of sR-I, attempting to arrive at a purified preparation. Several effective enrichment steps have been developed, including hydrophobic interaction chromatography, a high-yield lysolecithin extraction procedure and density gradient centrifugation. Enriched preparations were obtained, including an isolated fraction of halobacterial flagella.