Aggregated and Monomeric Bacteriorhodopsin Pumps Protons

Abstract

Bacteriorhodopsin (BR) molecules are arranged as immobilized trimers in the hexagonal crystalline lattice of the purple membrane. Experiments were designed to answer the question whether the hexagonal state of aggregation is required for proper functioning of this light-driven proton pump. Using the detergents Triton X-100 and octylglucoside BR was solubilized and incorporated as monomers into large unilamellar lipid vesicles. The thermotropic behaviour of these vesicles, i. e. of the lipid phase and of the protein-protein interactions, was monitored by means of the fluorescence polarization probe DPH and circular dichroism (CD) measurements, respectively. Light-induced pH-changes of vesicle suspensions were observed with a pH-electrode. The presence of exciton coupling features in the visible CD spectrum of aggregated BR and its absence in monomeric BR allow the determination of its aggregation state. In accordance with previous work1 the results indicate that at temperatures below the lipid phase transition, BR crystallizes into patches with the same hexagonal lattice as is observed in purple membrane, whereas above the transition the lattice disaggregates and the BR molecules are monomeric. In DMPC-BR vesicles the CD transition curve caused by changes in the aggregation state of BR is shifted by about 6–8°C towards lower temperatures in comparison to the lipid phase transition. The midpoint of the latter transition (about 23°C) was not changed due to the presence of BR. Illumination resulted in an alkalization of the external medium independently of the BR aggregation state.