Peptide:Lipid Ratio and Membrane Surface Charge Modulate the Mechanism of Action of the Antimicrobial Peptide BP100

Abstract

We present a comprehensive study of the conformational and functional properties of BP100 (H-KKLFKKILKYL-NH2) upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of zwitterionic PC and negatively charged PG. Theoretical prediction and calculation of hydrophobic moment indicate that BP100 is a surface-seeking amphipathic helix. Accordingly, CD spectra showed that, upon binding to PG-containing LUVs, BP100 acquires α-helical conformation. NMR data showed that the helix spans residues 3-11. Moreover, CD spectra evinced peptide aggregation in the membrane and/or vesicle aggregation, as a function of peptide:lipid ratio and PG content. Dynamic light scattering confirmed vesicle aggregation under conditions of electroneutralization. BP100 decreased the absolute value of the zeta (ζ)potential of LUVs with lower PGcontents. For higher PG contents, the ζ potential remained initially constant, a decrease occurring when ca. 80% of the negatively charged lipids were neutralized. This effect was rationalized in terms of an ion exchange process. Higher peptide concentrations were required for leakage from LUVs aqueous inner compartment at high ionic strength. Moreover, while a gradual release took place at low peptide:lipid ratios, instantaneous lossoccurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs also evinced BP100-promoted disruption of negatively charged membranes. We propose that the mechanism of action of BP100 is a function of both peptide:lipid ratio and negatively charged lipid content. While the gradual release of inner contents is a consequence of membrane perturbation by a small number of peptide molecules (monomers or small aggregates), membrane disruption results from a sequence of events: peptide aggregation on the membrane surface, leading to lipid clustering, andgiving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.