Direct transmembraneous reconstitution of bacteriorhodopsin into planar phospholipid bilayers

Abstract

A new method of incorporating bacteriorhodopsin molecules into planar lipid bilayers was developed and compared with a conventional system where purple membranes were adsorbed onto planar bilayers. By the new method, purple membrane sheets were first solubilized with a detergent and the solubilized fraction was directly added to an aqueous phase of a preformed planar bilayer membranes. Then, the solubilized bacteriorhodopsin molecules were spontaneously incorporated into the planar bilayers. Upon illumination, a steady state electric current was generated and the magnitude of the current was hardly affected by the presence of an uncoupler, FCCP. Furthermore, when bacteriorhodopsin molecules were incorporated into planar bilayers in high temperature range, a transient capacitive peak current was mostly eliminated by successive perfusions with a buffer at room temperature. These properties are in contrast with those of conventional method and strongly indicate that the bacteriorhodopsin molecules are transmembraneously reconstituted into planar bilayer membranes by the new method. The new method will prove useful in quantitative investigation of the properties of active ion transport.