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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Fluctuations and the Rate-Limiting Step ofPeptide-Induced Membrane LeakageClaudia Mazzuca 1 , Barbara Orioni 1 , Massimiliano Coletta 2 ,Fernando Formaggio 3 , Claudio Toniolo 3 , Giuseppe Maulucci 4 ,Marco De Spirito 4 , Basilio Pispisa 1 , Mariano Venanzi 1 ,and Lorenzo Stella 11 Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata",Rome, 00133, Italy; 1 Dipartimento di Medicina Sperimentale e Scienze Biochimiche,Università di Roma "Tor Vergata", Rome, 00133, Italy; 3 Dipartimento di ScienzeChimiche, Università di Padova, Padova, 35131, Italy; 4 Istituto di Fisica,Università Cattolica del Sacro Cuore, Rome, 00168, ItalyIntroductionPeptide-induced vesicle leakage is a common experimental test for the membraneperturbingactivity of antimicrobial peptides. Obviously, a quantitative analysis of thepeptide-induced leakage kinetics can potentially provide several insights into themechanism of pore formation. The release curves determined in these experiments in mostcases share two peculiar characteristics: they are usually very slow, requiring minutes tohours for complete release of vesicle contents, and exhibit a biphasic behavior (Figure 1).In this work, we systematically evaluate all of the possibilities for the rate-limiting step, bytaking as an a example a very well characterized peptide, i.e. the peptaibol trichogin GA IV[1-4], and provide a stochastic model of the leakage process based on the discrete nature ofa vesicle suspension.Results and DiscussionStopped-flow kinetic experiments show that peptide-membrane binding, peptidetranslocation to the inner leaflet of the membrane and peptide aggregation in the bilayer arecompleted in a few seconds. Direct observation of peptide-induced GUV leakage indicatesthat, once a pore is formed, liposomes empty very quickly. Therefore, all these processescan be ruled out as the rate-limiting step of the vesicle leakage process, which is muchslower. On the other hand, our data show that vesicle leakage rate increases with vesicleradius, and peptides are continuously and rapidly exchanging among vesicles. This led usto propose a simple stochastic model: a vesicle (or cell) suspension is obviously a discretesystem, and the release of the contents of each vesicle is not influenced by the otherliposomes in the sample. Furthermore, a liposome is a nanoscopic system, and the number1Released fraction0.500 200 400 600 800 1000 1200Time (s)Fig. 1. Kinetics of carboxyfluorescein release after addition of the trichogin GA IVanalogue F10 [1] to a LUV suspension. [Lipid]=0.2mM and [F10]=1.2 μM; 2.3 μM; 3.7μM; 4.7 μM; 6.9 μM; 10.8 μM; (from bottom to top). The dashed lines are the best fit tothe data with the model described in the present paper.368