Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition

Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aqueous-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phases, and ternary phase-separated membrane compositions. Fluorescence lifetime correlation spectroscopy was used to determine the lateral mobility of the lipid and the protein, and electrochemical impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have a profound impact on membrane electrochemical resistance, where the resistance of a ternary symmetric phase-separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl--glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 times faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that the interplay of lipid packing across both membrane leaflets and the concentration of GM1 both affect the extent of cholera toxin aggregation and the consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity to build greater biomimicry into biophysical models, and the approach described demonstrates the value of MSLBs in studying aggregation and the membrane-associated multivalent interactions prevalent in many carbohydrate-mediated processes.