Transition between Different Diffusion Modes of Individual Lipids during the Membrane‐Specific Action of As‐CATH4 Peptides

Abstract The cell membrane permeabilization ability of immune defense antimicrobial peptides (AMPs) is widely applied in biomedicine. Although the mechanisms of peptide–membrane interactions have been widely investigated, analyses at the molecular level are still lacking. Herein, the membrane‐specific action of a native AMP, As‐CATH4, is investigated using a single‐lipid tracking method in combination with live cell and model membrane assays conducted at different scales. The peptide–membrane interaction process is characterized by analyzing single‐lipid diffusion behaviors. As‐CATH4 exhibits potent antimicrobial activity through bacterial membrane permeabilization, with moderate cytotoxicity against mammalian cells. In‐plane diffusion analyses of individual lipids show that the lipid molecules exhibit non‐Gaussian and heterogeneous diffusion behaviors in both pristine and peptide‐treated membranes, which can be decomposed into two Gaussian subgroups corresponding to normal‐ and slow‐diffusive lipids. Assessment of the temporal evolution of these two diffusion modes of lipids reveal that the peptide action states of As‐CATH4 include surface binding, transmembrane defect formation, and dynamic equilibrium. The action mechanisms of As‐CATH4 at varying concentrations and against different membranes are distinguished. This work resolves the simultaneous mixed diffusion mechanisms of single lipids in biomimetic cell membranes, especially during dynamic membrane permeabilization by AMPs.