Advanced Models of Biological Membranes with Hydrogenous and Deuterated Natural Glycerophospholipid Mixtures

Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simpler model systems including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detec- tor (HPLC-ELSD) enabled a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. For this study, we produced and employed phoshatidyl- choline (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bi- layers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very com- parable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.