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Aggregated peptides per vesicle50025000 1 2Pores per vesicleFig. 2. Correlation between the average number of pores in a single vesicle, obtained fromthe fit to the leakage kinetics, and the number of membrane-bound peptide moleculesparticipating in aggregates, determined from time-resolved fluorescence experiments atequilibrium. ePC/cholesterol LUVs, [lipid]=0.2 mM, vesicle radius 50 nm.of peptides or the number of pores in each vesicle can be rather small, well below thethermodynamic limit. Therefore, peptide exchange among vesicles produces significantfluctuations over time in the number of peptide molecules bound to each vesicle, and in theformation of pores.According to this model, the fast initial leakage is caused by those vesicles which,after the random distribution of peptides among liposomes, already contain at least onepore, while the slower release is associated to the time needed in an intact vesicle tooccasionally reach the critical number of bound peptides necessary for pore formation.Fluctuations due to peptide exchange among vesicles represent therefore the rate-limitingstep of such a slow mechanism. Such a model can be described quantitatively by thefollowing equation:R(t)= 1−ee−n[1−exp(−t/ ϕ )] −nt/τwhere R is the fraction of vesicles’ contents released, φ and τ are characteristic times forvesicle emptying through a formed pore, and for peptide exchange among vesicles,respectively, n is the average number of pores per vesicle, and t is time.Figure 1 reports the best fit of the leakage data to this equation as dotted lines. Themost important result of this analysis is that it provides an estimate of n , which in this casecould be compared with the concentration of membrane-bound peptides participating inaggregates, determined independently from time-resolved experiments [1]. The strongcorrelation between the two datasets (Figure 2) provides strong support both to the kineticmodel and to the hypothesis that aggregates of membrane-bound peptides constitute thepores.AcknowledgmentsThis work was supported by grant PRIN 2008 (Italian Ministry of Education, University and Research,MIUR).References1. Stella, L., et al. Biophys. J. 86, 936-945 (2004).2. Mazzuca, C., et al. Biophys. J. 88, 3411-3421 (2005).3. Gatto, E. et al. J. Phys. Chem. B 110, 22813-22818 (2006).4. Bocchinfuso, G. et al. J. Pept. Sci. 15, 550-558 (2009).369