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.
Department of Applied Physics, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603 Japan.
Yasunori
Y
Yokoyama
Lumi
L
Negishi
Taku
T
Kitoh
Masashi
M
Sonoyama
Yasuo
Y
Asami
Shigeki
S
Mitaku
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002151:Calorimetry; D002942:Circular Dichroism; D008081:Liposomes / Q000737:chemistry*; D044367:Phase Transition; D010713:Phosphatidylcholines / Q000737:chemistry*; D038761:Photobleaching; D013056:Spectrophotometry, Ultraviolet; D044366:Transition Temperature
D008081:Liposomes; D010713:Phosphatidylcholines; D001436:Bacteriorhodopsins