LAUSR

EPR spectra at 94 GHz or higher are sensitive to lipid lateral ordering, which provides key information on the structure and dynamics of biological membranes as well as protein–lipid interactions. High-frequency high-field EPR improves spectral resolution through increased g-factor resolution, enabling the determination of the motionally averaged gxx–gyy anisotropy, which reflects lipid lateral ordering. Lipid rafts are domains of plasma membranes enriched with cholesterol and sphingolipids and are essential for cellular functions. Here, we investigated the lateral ordering of lipid rafts using high-field EPR. The analyses reveal that lateral ordering is more sensitive to raft-domain formation than the conventional order parameter. Lateral ordering is a sensitive parameter for both lipid phase transition and cholesterol/sphingomyelin content. Our data also show that raft and nonraft domains exhibit distinct lateral order profiles across the lipid bilayer. In addition, we characterized the membrane interaction and peptide-induced lipid-lateral-ordering changes of an antibacterial γ-AApeptide (AA1). The results illustrate that AA1 interacts strongly with bacterial membranes, induces significant lipid lateral ordering and membrane thinning, and subsequently disrupts bacterial membranes. Our findings suggest that AA1 interacts with and disrupts bacterial membranes through a carpet-like mechanism. Taken together, these results highlight the sensitivity of high-field EPR for elucidating membrane order and dynamics as well as protein–lipid interactions.