Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers.

ABSTRACT

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.