In Situ Monitoring of Phospholipid Flip-Flop via Fluorescence Self-quenching

Many antimicrobial peptides (AMPs) exert their activity by disrupting the integrity of the bacterial plasma membrane. One of the membrane-disrupting mechanisms of AMPs involves the formation of toroidal pores, composed of α-helices and lipid headgroups. These pores enable the diffusion of lipid molecules to the opposite leaflet without exposing their headgroups to the hydrocarbon core. Consequently, an increase in lipid transbilayer diffusion (flip-flop) in the presence of AMPs is an important characteristic for AMP structure and function. However, real-time monitoring of the rapid flip-flop in the presence of transmembrane pores has not been achieved because of the permeation of membrane-impermeable reagents and/or the low time resolution of the conventional assays. Herein, we have developed a fluorescence quenching-based flip-flop assay. The flip-flop rates obtained by our method were consistent with those measured by the conventional dithionite reduction assay, confirming the reliability of our approach. The real-time monitoring of the flip-flop process in the presence of the AMP, magainin 2, using the self-quenching assay suggested that the disordered toroidal pores composed of 2 magainin molecules facilitate flip-flop. The newly developed assay will provide a better understanding of the interactions between AMPs and lipid bilayers.