The EcCLC antiporter embedded in lipidic liquid crystalline films – molecular dynamics simulations and electrochemical methods

Abstract

Lipidic-liquid crystalline nanostructures (lipidic cubic phases), which are biomimetic and stable in an excess of water, were used as a convenient environment to investigate the transport properties of the membrane antiporter E. coli CLC-1 (EcCLC). The chloride ion transfer by EcCLC was studied by all-atom molecular dynamics simulations combined with electrochemical methods at pH 7 and pH 5. The cubic phase film was used as the membrane between the chloride donor and receiving compartments and it was placed on the glassy carbon electrode and immersed in the chloride solution. Structural characterization of lipidic mesoscopic systems with and without the incorporation of EcCLC was performed using small-angle X-ray scattering. The EcCLC transported chloride ions more efficiently at more acidic pH, and the resistance of the film decreased at lower pH. 4,4-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) employed as an inhibitor of the protein was shown to decrease the transport efficiency upon hydrolysis to DADS at both pH 7 and pH 5. The molecular dynamics simulations, performed for the first time in lipidic cubic phases for EcCLC, allowed studying the collective movements of chloride ions which can help in elucidating the mechanism of transporting the ions by the EcCLC antiporter. The protein modified lipidic cubic phase film is a convenient and simple system for screening potential inhibitors of integral membrane proteins, as demonstrated by the example of the EcCLC antiporter. The use of lipidic cubic phases may also be important for the further development of new electrochemical sensors for membrane proteins and enzyme electrodes.