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

Phospholipid bilayers formed at solid-liquid interfaces have garneredinterest as mimics of cell membranes to model association reactions of proteins withlipid bilayer-tethered ligands. Despite the importance of understanding how liganddensity in a lipid bilayer impacts the protein-ligand association response, relating theligand-modified lipid fraction to the absolute density of solution-accessible ligands in alipid bilayer remains a challenge in interfacial quantitative analysis. In this work,confocal Raman microscopy is employed to quantify the association of anti-biotin IgGwith 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 silicasurfaces. We examine the question of whether inter-leaflet lipid translocationcontributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparingtheir protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding ofthe antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation ofIgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the similar to 60%greater anti-biotin capture observed atfluid-phase bilayers compared tofluid-phase monolayers prepared at equivalent biotinfractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximalleaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipidflip-flop, a facile process influid-phasesupported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed influid-phase phospholipid bilayers.

Automated summary

This study investigates the impact of ligand density on the protein-ligand association response in phospholipid bilayers formed at solid-liquid interfaces. Confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with biotinylated lipids dispersed in gel-phase or liquid-crystalline supported lipid bilayers. The results suggest caution in interpreting quantitative studies of protein binding to lipid-tethered ligands in fluid-phase phospholipid bilayers.