Quantitative single-vesicle analysis of antimicrobial peptide-induced leakage

Although the research field of antimicrobial peptides has attracted considerable scientific attention in the past decades, the microbicidal mechanisms of antimicrobial peptides still remain elusive. One of the keys to a more profound comprehension of the function of these peptides is a deeper understanding of their interactions with phospholipid membranes. In this study, the membrane-permeabilizing effects of antimicrobial peptides were scrutinized by combining two biophysical techniques. Confocal fluorescence microscopy to visualize leakage from individual surface-immobilized lipid vesicles was combined with fluorescence correlation spectroscopy to quantify leakage from a bulk collection of lipid vesicles in aqueous solution. Quantitative correlation between the two techniques was achieved through a detailed experimental protocol. The potential of combining the two techniques was tested using three canonical antimicrobial peptides: melittin, magainin 2, and mastoparan X. The results demonstrate an unprecedented level of insight into the molecular processes governing antimicrobial peptide-induced permeabilization of phospholipid membranes.