The Interaction Between the Antimicrobial Peptide K-Hya1 and Model Membranes: Distinct Action in Neutral or Negatively Charged Bilayers

Abstract

Antimicrobial peptides (AMPs) are important molecules for the innate immune defense system of plants and animals. The antimicrobial peptide Hylina1 (Hya1) was isolated from arboreal South African frog Hypsiboas albopunctatus. In this work, we investigate the interaction of an analogue peptide, K-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with model membranes composed of DPPC (dipalmitoyl phosphatidylcholine), DPPG dipalmitoyl phosphatidylglycerol) and DPPC:DPPG 1:1 or of POPC (palmitoyl oleoyl phosphatidylcholine) and POPC:POPG (palmitoyl oleoyl phosphatidylglycerol) with different techniques. Differential Scanning Calorimetry (DSC) profiles show distinct environments in the bilayer with anionic lipids, suggesting a disturbed region due to peptide adsorption, and an unaltered region. On the other hand, in neutral membranes an average perturbation is observed, which suggests a superficial interaction. Steady-state fluorescence spectroscopy of the intrinsic Trp residue shows a deeper insertion of this residue into charged bilayers as compared with neutral membranes. Dye-leakage experiments show that membrane charge also modulates the kinetics of membrane permeabilization, which is much faster for charged bilayers. Optical microscopy of giant unilamellar vesicles (GUVs) in the fluid phase revealed a different mechanism of action of the peptide in the presence or absence of negatively charged lipids. K-Hya1 induces small perturbations in POPC vesicles, causing membrane permeabilization without morphological changes. On the other hand, the peptide induces permeabilization accompanied by a large increase in surface area of POPC:POPG vesicles, suggesting the opening of several pores in the bilayer. Taken together, the results clearly show a peptide-bilayer interaction modulated by the presence of negatively charged lipids. Data can be interpreted as a different orientation of the peptide in the bilayer: parallel to the surface in neutral membranes and stable and crossed in anionic membranes.