Translocase Activity and Asymmetric Model Membranes Probed by Neutron Scattering

Abstract

Biological membranes are almost universally asymmetric with individual leaflets containing different lipid species at varying concentrations. This asymmetry may play a key role in biological functions such as protein-membrane interactions, membrane trafficking, and cellular signaling. Traditional model membrane systems are comprised of symmetric membrane leaflets and though progress has been made in developing asymmetric model membranes, these systems are unstable and easily contaminated. We have developed an actively asymmetric biomembrane model system by purifying and reconstituting the well-characterized E. coli phospholipid ABC transporter MsbA into proteoliposomes that mimic the phospholipid composition of a physiological bacterial membrane. Subsequent MsbA-mediated phospholipid translocation and resultant membrane asymmetry was studied using small-angle neutron scattering (SANS). SANS is exceptionally well suited for studying the phospholipid distribution perpendicular to the bilayer plane due to its nondestructive nature and sub-nanometer resolution. Additionally, the hydrogen/deuterium (H/D) neutron scattering contrast variation from isotopic phospholipid labeling enables us to track the translocation of lipid membrane components without introducing bulky fluorescence or spin labels. In this study, the MsbA-mediated translocation of the phospholipid phosphatidylethamolamine (PE) between proteoliposome leaflets and the formation of asymmetric bilayers was studied using SANS for the first time. The sustained asymmetry in these model protocells will be central for future studies on biomimetic membrane systems and their role in diverse membrane-directed biological processes.