Mechanisms Of Antimicrobial Peptide Action Determined Using Chemical And Collisional Quenching Assays

Abstract

Peptide-lipid interactions are pertinent to antimicrobial peptides (AMP) activity, stringency, and selectivity. The ability of AMPs to disrupt the target cell's lipid bilayer has been described using the nonspecific “carpet” model or using models that assume stable transbilayer pores (e.g. barrel stave pores). With the carpet model, there is a strong electrostatic and hydrophobic attraction between peptide and the lipid interfacial groups such that the peptides blanket the cell membrane. Carpet model peptides can then kill the organism by disrupting the lipid bilayer and causing loss of cellular contents. The stable pore model consists of fixed transmembrane structures that rely on an amphipathic amino acid sequence to form membrane-spanning pores. Included with these models, peptides can also induce flip-flop of bilayer lipids or form transient pores. We have developed chemical and collisional quenching assays that help determine the mode of lipid disruption associated with naturally occurring and synthetically designed AMPs. The assays require large unilamellar vesicles (LUVs) with fluorophore-attached lipid head groups in both inner and outer leaflets of the bilayer. Only peptides with stable pore-forming or detergent-like activity allow quenchers access to the inner leaflet. The combination of these quenching assays with leakage experiments and cryo-electron microscopy allows for a more complete description of the mechanism of membrane disruption by peptides.