Real-time quantitative analysis of lipid disordering by aurein 1.2 during membrane adsorption, destabilisation and lysis

Abstract

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.

Melittin peptides exhibit different activity on different cells and model membranes

“…Although the mechanism of AMP action is unknown and pore formation in cell membranes may be responsible for lytic ability, it is well established that AMP interact directly with the cell membrane surface and are able to enter the lipid bilayer, resulting in cell membrane disruption and leakage of cytoplasmic components which leads to killing of microbes (Lee et al 2010;Izadpanah and Gallo 2005;Lorenzon et al 2012;Sani et al 2013). AMP have attracted much attention as an alternative to antibiotics because of their effect against bacterial infections and drug resistant bacteria (Juba et al 2013;AnayaLopez et al 2013).…”

Development of a μO-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

“…The mass of an adsorbed molecular layer was calculated with the use of the De Feijter formula (42) where the dn/dc values of 0.135 and 0.182 ml/g were used for calculating the adsorbed lipids and peptides, respectively (43,44). Birefringence (⌬n f ) was obtained by calculating the difference between two effective refractive indices, namely refractive index of transverse magnetic (TM) waveguide mode (n TM ) and refractive index of transverse electric (TE) waveguide mode (n TE ) (43)(44)(45). The respective effective adlayers n TM and n TE corresponding to the measured TM and TE phase changes are calculated by fitting data to a waveguide equation (44 -46).…”

Effects of linear amphiphilicity on membrane interactions of C-terminal thrombin peptides

“…The birefringence (∆n f ), obtained from the refractive indices for the TM and TE waveguide modes (assuming the bilayer thickness to be constant), reflects ordering of the lipid molecules in the bilayer, and decreases with increasing disordering of the bilayer (26,27). Consequently, ∆n f can be used to monitor ordering transitions in lipid bilayers as a result of peptide binding and incorporation, and therefore offers a simpler alternative to, e.g., order parameter analyses in 2 H-NMR spectroscopy (28).…”