THE RECONSTITUTION OF PURPLE MEMBRANES FR OM DELIPIDATED BACTERIORHODOPSIN USING ENDOGENOUS AND EXOGENOUS LIPIDS - THE EFFECT OF STRUCTURALAND EXTERNAL FACTORS

The effect of lipid environments and external factors (such as tempera ture and ionic strength of aqueous medium) on 2D-protein crystal forma tion was studied in reconstituted membranes prepared from delipidated bacteriorhodopsin mixed with total polar lipid fraction (extracted fro m Halobacterium halobium cells), egg yolk phosphatidylcholine, and dip almitoylphosphatidylcholine. Reconstitution was performed at lipid-pro tein ratios close to their ratio in the native purple membrane. The re constituted membranes were characterized by the data of absorption spe ctroscopy, visible CD, freeze-fracture electron microscopy, and DSC. D epending on the lipid structure and the lipid-protein ratio, various m odifications of the purple membrane were obtained differing in the cha racter and the type of bacteriorhodopsin packing. The crystal structur e of bacteriorhodopsin can be formed by its. incorporation into the en dogenous lipid environment at the native lipid-protein ratio (0,33/1) with low NaCl concentration of the aqueous buffer. The regeneration of the crystal structure under an exess of endogenous lipids requires hi gh NaCl concentration (3,5-4 M) or heating up to 40-45 degrees C. Tn t he case of egg phosphatidylcholine, highly ordered protein structures are observed only at the high NaCl concentrations. In the membrane rec onstituted with the endogenous lipids, bacteriorhodopsin oligomers pac k in hexagonal lattice of p3 symmetry, while in the system containing egg phosphatidylcholine the protein crystallizes in orthogonal lattice of p22(1)2(1) symmetry. The protein ordering proceeding in the membra ne seems to depend to the certain extent on the electrostatic surface potential and interface permittivity which are influenced by the delic ate balance of electrostatic interactions between protein, polar heads of lipid molecules, and ions in the aqueous media of the membrane sur face.