Ampullosporin A is an antimicrobial, neuroleptic peptaibol, the behavior of which was investigated in different membrane mimetic environments made of egg yolk L-α-phosphatidylcholine. In monolayers, the peptaibol adopted a mixed α/310-helical structure with an in-plane orientation. The binding step was followed by the peptide insertion into the lipid monolayer core. The relevance of the inner lipid leaflet nature was studied by comparing ampullosporin binding on a hybrid bilayer, in which this leaflet was a rigid alkane layer, and on supported fluid lipid bilayers. The membrane binding was examined by surface plasmon resonance spectroscopy and the effect on lipid dynamics was explored using fluorescence recovery after photobleaching. In the absence of voltage and at low concentration, ampullosporin A substantially adsorbed onto lipid surfaces and its interaction with biomimetic models was strongly modified depending on the inner leaflet structure. At high concentration, ampullosporin A addition led to the lipid bilayers disruption. 1. Introduction Living cells naturally produce diverse membranotropic peptides displaying antifungal and/or antibacterial activities. These properties have been attributed to their molecular interaction with the target cell membranes. Three basic models have been proposed for membrane interaction mechanisms leading to membrane permeabilization. The first model consists in the formation of barrel-stave pores [1], in which the pore lumen is formed by the polar side of amphipathic and helical peptide monomers assembled into a bundle. The second major mechanism that has been proposed for membranolytic, amphipathic peptides is the formation of the so-called “toroidal” transient pores [2, 3]. In this model, the peptides bind to the polar head groups of the cell membrane phospholipids (PL) and, above a critical peptide concentration, continuously bend the lipid leaflet favouring the formation of a pore lined by both the peptides and the lipid polar headgroups. The third major model is called the “carpet-like” mechanism. While the two former modes of action require the formation of a pore structure, the latter one is subsequent to the accumulation of flat-oriented peptides at the lipid bilayer surface and may lead to membrane disruption and formation of mixed lipid-peptide aggregates [4]. The peptaibiotics’ family [5, 6] comprises a large number of defense peptides, most of which share common features such as an acetylated N-terminal amino acid residue, a C-terminal amino alcohol and a high proportion of nonproteogenic residues such
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