Heterogeneous Structural Disturbance of Cell Membrane by Peptides with Modulated Hydrophobic Properties

Extensive effort has been devoted to developing new clinical therapies based on membrane-active peptides (MAPs). Previous models on the membrane action mechanisms of these peptides mostly focused on the MAP–membrane interactions in a local region, while the influence of the spatial heterogeneity of the MAP distribution on the membrane was much ignored. Herein, three types of natural peptide variants, AS4-1, AS4-5, and AS4-9, with similar amphiphilic α-helical structures but distinct hydrophobic degrees (AS4-1 < AS4-5 < AS4-9) and net charges (+9 vs. +7 vs. +5), were used to interact with a mixed phosphatidylcholine (PC) and phosphatidylglycerol (PG) membrane. A combination of giant unilamellar vesicle (GUV) leakage assays, atomic force microscopy (AFM) characterizations, and molecular dynamics (MD) simulations demonstrated the coexistence of multiple action mechanisms of peptides on a membrane, probably due to the spatially heterogeneous distribution of peptides on the membrane surface. Specifically, the most hydrophobic peptide (i.e., AS4-9) had the strongest membrane binding, perturbation, and permeabilization effects, leading to the formation of large peptide–lipid aggregates (10 ± 5 nm in height and 150 ± 50 nm in size), as well as continuous fragments and ridges on the supported membrane surface. The AS4-5 peptides, with a half-hydrophilic and half-hydrophobic structure, induced membrane lysis in addition to reconstruction. The most hydrophilic peptide AS4-1 only exhibited unstable binding on the supported membrane surface. These results demonstrate the heterogeneous structural disturbance of model cell membranes by amphiphilic α-helical peptides, which could be significantly strengthened by increasing the degree of hydrophobicity and/or local number density of peptides. This work provides support for the modulation of the membrane activity of MAPs by adjusting their hydrophobicity and local concentration