HOW MEMBRANE ORDER, ELECTROSTATICS, AND LIPID PHASE STATE GOVERN ANTIMICROBIAL PEPTIDE FUNCTION

Antimicrobial peptides (AMPs) act primarily on the lipid matrix of biological membranes, yet their selectivity cannot be explained solely by peptide charge or amphipathicity. Instead, membrane order, electrostatics, lipid composition, and phase state define the physical landscape governing peptide binding, insertion, lipid-packing perturbation, and membrane disruption. Here, we establish a membrane-centered biophysical framework in which AMP activity emerges from the interplay between electrostatic and mechanical properties of the target membrane. Using model systems ranging from Langmuir monolayers to large and giant unilamellar vesicles, combined with spectroscopy, ζ-potential measurements, calorimetry, and molecular dynamics simulations, we investigated the membrane interactions of natural and synthetic amphipathic peptides. Our results reveal that these peptides behave as sensors of membrane physicochemical state rather than indiscriminate membrane disruptors. Peptide activity is favored by lipid disorder and phase boundaries, whereas highly ordered and sterol-rich membranes restrict productive insertion and disruption. Moreover, distinct membrane compositions—including phosphatidylserine exposure, PE/PG mixtures, and hopanoid-containing membranes—modulate peptide activity through coupled, non-additive effects on membrane electrostatics, packing, and elasticity. Together, these findings demonstrate that antimicrobial and anticancer activities arise from the ability of peptides to recognize and exploit specific physicochemical signatures of target membranes. These results demonstrate that antimicrobial and anticancer activities emerge from the ability of peptides to sense and exploit physicochemical signatures of target membranes, providing general principles for the rational design of selective membrane-active peptides.