Reconstitution of bacteriorhodopsin into cyclic lipid vesicles.

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.

Photobleaching of bacteriorhodopsin solubilized with triton X-100.

In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.

Effect of lipid phase transition on molecular assembly and structural stability of bacteriorhodopsin reconstituted into phosphatidylcholine liposomes with different acyl-chain lengths.

Previous studies on the correlation between bacteriorhodopsin (bR) disassembly and photobleaching suggested that a weakening of intermolecular interactions is responsible for irreversible photobleaching (Mukai, Y.; Kamo, N.; Mitaku, S. Protein Eng. 1999, 12, 755-759; Yokoyama, Y.; Sonoyama, M.; Mitaku, S. J. Biochem. 2002, 131, 785-790). In order to reveal the role of the lipid matrix in bR assembly and photobleaching, we reconstituted bR into diacylphosphatidylcholine (diacylPC) vesicles with three different saturated acyl-chain lengths. Visible circular dichroism (CD) spectra collected upon photobleaching showed an exciton-to-positive transition for bR reconstituted into dimyristoyl-, dipalmitoyl-, and distearoyl-PC vesicles around 17, 35, and 50 °C, respectively. These transition temperatures were close to the main transition temperature of reconstituted vesicles measured by calorimetry, indicating that the lipid phase transition brought about protein disaggregation. Absorption spectra of reconstituted bR exhibited a blue-shifted retinal absorption during protein disaggregation in the ground state. Absorption spectra collected from samples exposed to continuous illumination revealed an accumulation of M-intermediate state, and the absorption band around 410 nm underwent a blue shift through the visible CD change, indicating conformational perturbations due to protein disassembly. Irreversible photobleaching started to occur at the same temperature range as the change in the visible CD spectrum, clarifying the correlation between bR disassembly and photobleaching. In contrast, no thermal bleaching was observed below 60 °C for any sample kept in the dark. A plausible model for irreversible photobleaching is presented, on the basis of these experimental results.