Atomic Force Microscopy and Electrochemical Studies of Melittin Action on Lipid Bilayers Supported on Gold Electrodes

ABSTRACTMelittin is an amphipathic helical peptide which shows strong membranolytic activity against bacterial cells as well as human erythrocytes. It is often considered as a good model for general studies on interactions of antimicrobial peptides with biological membranes since the detailed mechanisms of their action on biological membranes are still subject of intense debate. In this paper we have used electrochemical methods combined with in situ AFM imaging in order to evaluate the mechanisms involved in melittin membranolytic activity. We have observed that high concentration of the peptide causes rapid degradation of a single component DMPC bilayer supported on gold electrode. The lipid membrane undergoes micellization and subsequent dissolution. This indicates that under such conditions melittin acts according to detergent-like mechanism. The mode of melittin action differs substantially when the peptide concentration is lowered. In this case, the changes in DMPC bilayer structure are less rapid and at the initial stages the peptide adsorbs on top of the membrane. This process is followed by fluidization of the DMPC film, which facilitates further reorientation and insertion of melittin into the bilayer. As a result, the permeability of the membrane is increased. AFM data shows that sharp differentiation between carpet and toroidal pore mechanism is difficult and it is very likely that melittin acts according to mixed mechanism. Yet different behavior of melittin was observed for the mixed DMPC/Cholesterol bilayer. The susceptibility of the membrane to melittin action was reduced probably due to the increased packing within the hydrocarbon chain region of the bilayer. We have also noticed that the fluidization of the membrane is a common feature for all systems studied here. Thus, it seems to be a crucial step for melittin action which enables the peptide molecules to adopt proper orientation either for pore formation or disruption of the membrane.