The Role of “Raft-Like” Membranes on Antimicrobial Peptide-Lipid Bilayer Interactions

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In vitro studies of antimicrobial peptides have largely focused on simple two-component vesicle systems consisting of two miscible lipids. However, real membranes are vastly more complex. In this study, we investigate the in vitro interaction between antimicrobial peptides and non-homogenous, cholesterol-containing model membranes with fluorescence-based and other biophysical assays. Phase mappings of ternary and binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyol-sn-glycero-3-phosphocholine (DPPC), and cholesterol have shown that separate phasal domains can exist in liposomes at a broad range of lipid compositions. The ability of these domains to form liquid-liquid coexisting phases, particularly liquid-disordered (ld)-liquid-ordered (lo) regions sometimes referred to as membrane “rafts”, raises the question of their stability or integrity in relation to similar cholesterol containing membranes. We therefore have studied the interactions of these membranes in response to antimicrobial peptide attack, using a series of antimicrobial peptides specifically chosen to disrupt membranes by a variety of mechanisms. We have found that liposomes containing these raft domains display far greater membrane disruption than similar homogenous model membranes containing equal mole percentages of cholesterol. Pyrene-PC translocation assays demonstrate an increased susceptibility of these raft-like membranes to form toroidal-pore-like structures in the bilayer. Binding parameters obtained from isothermal titration calorimetry measurements and circular dichroism spectroscopy indicate that preferential peptide-lipid binding interactions alone are not responsible for the differences in membrane disruption. These combined results indicate that an increase in local peptide concentration as a consequence of the peptide partitioning preferentially to ld domains allows for increased antimicrobial activity by pore formation or fragmentation of the membrane.