Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichiacoli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5muM), indicating a surface-associated state without significant perturbation in membrane structure. At 10muM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20muM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action.
The membrane destabilising properties of the antimicrobial peptides (AMP) aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, have been studied by dual polarisation interferometry (DPI). The overall process of peptide induced membrane destabilisation was examined by the changes in bilayer order as a function of membrane-bound peptide mass per unit area and revealed three different modes of action. Aurein 1.2 was the only peptide that significantly destabilised the neutral membrane (DMPC), while all four peptides induced destabilisation of the negatively charged membrane (DMPC/DMPG). On DMPC, citropin 1.1, maculatin 1.1 and caerin 1.1 bound irreversibly at low concentrations but caused a reversible drop in the bilayer order. In contrast to DMPC/DMPG, these three peptides caused a mass drop at the higher concentrations, which may correspond to insertion and bilayer expansion. The critical level of bound peptide necessary to induce membrane destabilisation (peptide:lipid ratio) was determined and correlated with peptide structure. As the most lytic peptide, aurein 1.2 adsorbed strongly prior to dissolution of the bilayer. In contrast, the binding of citropin 1.1, maculatin 1.1 and caerin 1.1 needed to reach a critical level prior to insertion into the membrane and incremental expansion and disruption. Our results demonstrate that sequential events can be monitored in real-time under fluidic conditions to elucidate the complex molecular mechanism of AMP action. In particular, the analysis of birefringence in real time allows the description of a detailed mechanistic model of the impact of peptides on the membrane bilayer order. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.
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