Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis.
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.
Lee
Tzong-Hsien
TH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Heng
Christine
C
Swann
Marcus J
MJ
Gehman
John D
JD
Separovic
Frances
F
Aguilar
Marie-Isabel
MI
D000327:Adsorption; D000465:Algorithms; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D007368:Interferometry / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013997:Time Factors
D023181:Antimicrobial Cationic Peptides; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; C506635:aurein 1.2 peptide; C002773:dimyristoylphosphatidylglycerol; D004134:Dimyristoylphosphatidylcholine