The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporins family, have been produced and their hemolytic and lipidbinding properties compared to those of the wild-type protein. A thermodynamical approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpydriven process that occurs with a high affinity constant (1.7 x 10 8 M -1 ). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, even though it is necessary for correct pore 2 formation. Membrane binding is achieved through two separate mechanisms, i.e.recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site.Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.3

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