The interaction of four antimicrobial peptides, including magainin 2, melittin, polymyxin B and an artificial
peptide V4, with phospholipid membranes was investigated by fluorescence correlation spectroscopy (FCS) and confocal
imaging. Fluorophore entrapping and labeled vesicles were used to quantitatively determine the extent of antimicrobial
peptides interacting with membrane mimics and unravel their different mechanisms of action. It is shown that at high peptide/
lipid ratio, magainin 2 and melittin form pores to induce comparable level of membrane permeation while polymyxin
B and V4 disrupt the membrane. Considering its low solubility, V4 is much more active than others. Due to the hydrophobic
interactions, melittin, polymyxin B and V4 all promote aggregation when they perform their function. These experiments
demonstrate the feasibility of mechanistic studies by an FCS based approach.