Proton translocation across bacteriorhodopsin containing solid supported lipid bilayers

Abstract

Bacteriorhodopsin (BR) was incorporated in solid supported lipid bilayers by fusion of reverse phase vesicles on chemisorbed monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE) on gold substrates. The passive electrical behavior of the artificial membranes was monitored by impedance spectroscopy in order to determine both the membrane resistances and capacitances and to guarantee reproducibility of the bilayer formation. Illumination of the BR containing solid supported lipid bilayers resulted in a transient photocurrent as expected from earlier experiments with black lipid membranes. The present preparation technique however is advantageous because of its long term stability up to 1 day without loss of BR activity and its easy handling. We investigated the dependence of the photocurrent on the BR content, lipid environment, pH, and a proton carrier using a common current amplifier. Maximum current densities were obtained in the presence of negatively charged lipids like 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) or 1-palmitoyl-2-sn-glycero-3-phosphoglycerol (POPG) at a pH of 6.4. Moreover it could be shown that the pump activity of reconstituted BR is insignificantly influenced by the capacitance of the first self-assembled DMPTE-monolayer on the gold electrodes. This may be explained by an incomplete fusion of BR containing vesicles on the hydrophobic surface. Carbonylcyanid-4-trifluoromethoxy-phenylhydrazone (FCCP), a membrane soluble proton translocator, increases the membrane conductance as well as the capacitance of the lipid bilayer that was derived either from impedance spectroscopy or evaluation of the time constants of the transient photocurrents. (C) 1997 Elsevier Science Ireland Ltd.