Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation.

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.