Solid-supported lipid membranes for protein incorporation

Abstract

Solid-supported model membranes are thought to be particularly useful for studies of membrane proteins, as they open routes to investigation of such systems with surface-sensitive characterization techniques. We discuss a membrane system based upon a synthetic lipid that consists of alkyl tails attached through a glycerol backbone to a thiol-terminated polyethylene oxide spacer. This chemistry permits grafting to gold films supported, e.g., by silicon substrates. Membranes based on this compound are particularly robust while being well suited to membrane protein incorporation because they allow the protein to occupy trans-membrane space. Bilayer formation is based on rapid solvent exchange to produce membranes which are electrochemically tight while containing solvent below the membrane. This method also allows the formation of mixed membranes, and the modification of the trans-membrane space using backfilling molecules. The system has been studied with a range of mutually supportive techniques, including electrochemical impedance spectroscopy (EIS), AFM, ellipsometry, and neutron reflectivity. The particular combination of electrochemical measurements and neutron reflection permits structural characterization of reconstituted proteins while simultaneously testing protein activity in this synthetic environment. We discuss here the membrane optimization, as well as early results in which we have exposed the membranes to an anthrax protective antigen, PA63, a pore-forming protein that interacts with two auxiliary proteins, Lethal Factor and Edema Factor. PA63 is believed to act as a chaperone to allow LF and EF to pass the membrane barrier. The crystal structure of PA63 in unknown, but experimental and theoretical results suggest it incorporates an unusually long pore channel, nearly twice as long as that found in α-hemolysin, a prediction which we are testing. The latter work is done in collaboration with USAMRIID and NCI (Fort Detrick).