Reconstitution of delipidated bacteriorhodopsin with endogenous polar lipids.

Delipidated bacteriorhodopsin has been reconstituted with endogenous polar lipids from Halobacterium halobium. The vesicle (diameter, 250-500 A) formed are very stable, relatively homogeneous in bacteriorhodopsin and lipid content, and almost optically clear; a minor turbid fraction can be separated by gel filtration. Bacteriorhodopsin in the reconstituted vesicles has an inside out orientation and, on illumination, translocates protons efficiently from the medium to the interior of the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, both the rate and the extent of light-dependent proton uptake by the vesicles are decreased 3-6- and 5-15-fold, respectively, depending on the salt in the assay medium. Both the stimulation by valinomycin and the proton-translocating activity are higher in NaCl than in KCl. Bacteriorhodopsin in these vesicles as in purple membrane, undergoes light adaptation as indicated by a red shift (7-8 nm) of the absorption maximum. At low pH, the absorption maximum of reconstituted protein shows a 50-nm red shift, possibly due to protonation of an ionizable group which interacts with the chromophore. The latter group appears to be accessible only from the external medium.