SANS Investigation of the Response of DMPC-DMPG Lipid Bilayers to Membrane-Active Peptides

Abstract

Membrane-active peptides disrupt the integrity of cell membranes and form transmembrane pores in model lipid bilayers. Alamethicin and melittin are two extremely well-characterized examples of membrane-active peptides that are known to undergo a concentration-dependent transition from a surface-adsorbed state to a state in which transmembrane pores are formed, resulting in the death of the target cell. The action of these peptides strongly depends on the composition of the lipid bilayer membrane. In particular, charged lipids and cholesterol are thought to drive the cellular specificity of the cytotoxicity of these membrane active peptides. Further, lipid rafts, enriched domains in multi-component membranes, can concentrate or exclude proteins and peptides associated with lipid bilayers. SANS with contrast variation was used to probe the response of small-unilamellar vesicles (SUVs) composed of mixtures of the neutral lipid DMPC with the charged lipid DMPG to the presence of alamethicin and melittin. SUVs made of chain-deuterated d54-DMPC and DMPG at a molar ratio of 7: 3 were studied in the absence and presence of the two peptides in H2O/D2O mixtures containing 90% D2O solution. The measurements in 90% D2O, which is at the match point of the readily available d54-DMPC, greatly enhances the scattering from the hydrogenated components and ensure maximum signal for any in-bilayer aggregates or an asymmetric distribution between the leaflets of the bilayer of hydrogenated material that may form. The SANS experiments were performed using the BIO-SANS of HFIR at ORNL, which provided excellent signal for the dilute SUVs solutions. We found in both alamethicin and melittin, the asymmetric distribution of two lipids between the two monolayers increases with the peptide concentration. Interestingly, melittin was found to produce a stronger effect than alamethicin.