Leaflet-Dependent Effect of Anionic Lipids on Membrane Insertion by Cationic Cell-Penetrating Peptides

Cationic membrane-permeating peptides can cross membranesunassistedby transmembrane protein machinery, and there is consensus that anioniclipids facilitate this process. Although membranes are asymmetricin lipid composition, investigations of the impact of anionic lipidson peptide-membrane insertion in model vesicles primarily usesymmetric anionic lipid distributions between bilayer leaflets. Here,we investigate the leaflet-specific influence of three anionic lipidheadgroups [phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol(PG)] on insertion into model membranes by three cationic membrane-permeatingpeptides (NAF-1(44-67), R6W3, and WWWK). We report that outer leaflet anionic lipids enhancedpeptide-membrane insertion for all peptides while inner leafletanionic lipids did not have a significant effect except in the caseof NAF-1(44-67) incubated with PA-containing vesicles.The insertion enhancement was headgroup-dependent for arginine-containingpeptides but not WWWK. These results provide significant new insightinto the potential role of membrane asymmetry in insertion of peptidesinto model membranes.