Study of the antibacterial peptide P8.1: Effect on anionic vesicles using spectroscopic techniques

Antimicrobial peptides (AMPs) are promising complements to antibiotics, yet their membrane-level actions remain incompletely understood. In this work, we characterized how the "de novo" cationic AMP P8.1 interacts with anionic lipid bilayers composed of DPPG (gel phase) or DLPG (fluid phase) using zeta potential, tryptophan and Laurdan fluorescence, Raman microscopy, and a carboxyfluorescein (CF) leakage assay. P8.1 bound both lipids electrostatically, reduced zeta potential, and increased large unilamellar vesicles (LUVs) size. Binding kinetics were faster on DPPG multilamellar vesicles, whereas Trp fluorescence assays showed deeper insertion in DLPG (larger Trp blue-shift and lower acrylamide quenching). Laurdan generalized polarization (GP) increased in DLPG but not in DPPG, indicating reduced water access and higher local order in fluid bilayers. Raman spectra revealed diminished phosphate-band intensity in both systems and, in DLPG, a decreased gauche/trans ratio and narrower 1300 cm^-1 band consistent with tighter acyl-chain packing. Difference spectra further showed an amide I shift of P8.1, supporting a random-coil to α-helix transition upon binding to lipids. Finally, P8.1 induced ∼80 % CF leakage in DLPG LUVs within minutes. Together, the data indicate that P8.1 engages phosphate groups of lipids and then modulates bilayer structure in a phase- and mechanics-dependent manner-rigidifying short-chain, fluid DLPG and minimally perturbing gel-phase DPPG-providing mechanistic insight relevant to antibacterial activity.