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Fig. 2. Left: CD spectra in methanol of tylopeptin B and the TOAC-containing analogues(peptide concentration 10 -4 M). Right: Peptide-induced carboxyfluorescein (CF) leakage fromegg phosphatidylcholine /cholesterol (7:3) SUV for different [peptide]/[lipid] ratios (R -1 ).The TOAC spin-labelled analogues of tylopeptin B were submitted to a preliminaryconformational analysis by circular dichroism (CD) in different solvents. Recently, we haveshown that tylopeptin B is largely helical in solution, with a preference for the α- or the3 10 -helix type, depending upon the nature of the solvent [4]. The CD spectra of the spinlabelledanalogues (Figure 2, left, shows two representative examples) in methanol,2,2,2-trifluoroethanol and 30 mM sodium dodecylsulphate closely resemble those of theparent peptide, showing that in membrane-mimicking enviroments these peptides adopt anamphiphilic secondary structure.The membrane-modifying properties of these tylopeptin analogues were compared tothose of the parent peptide and trichogin GA IV (our standard membrane-activepeptaibiotic) by measuring the induced leakage of CF entrapped in small unilamellarvesicles (SUV). As shown in Figure 2 (right), [TOAC 13 ]tylopeptin and [TOAC 8 ]tylopeptinexhibited a very high activity in this membrane permeabilization assay that is comparableto or even slightly higher than that of the natural peptide.In summary, TOAC spin-labelled analogues of tylopeptin B can be considered goodmodels to investigate the detailed mechanism of membrane permeabilization by mediumlengthpeptaibiotics, using a combination of ESR techniques.AcknowledgmentsThis work was supported by Italian Ministry of Education, University and Research (PRIN 2008).References1. Marsh, D., Jost, M., Peggion, C., Toniolo, C. Biophys. J. 92, 473-481 (2007).2. Milov, A.D., Samoilova, M.I., Tsvetskov, J., Jost, M., Peggion, C., Formaggio, F., Crisma, M.,Toniolo, C., Handgraaf, J.-W., Rapp, J. Chem. Biodivers. 4, 1275-1297 (2007).3. Monaco, V., Formaggio, F., Crisma, M., Toniolo, C., Hanson, P., Milllhauser, G.L. Biopolymers 50,239-253 (1999).4. Gobbo, M., Poloni, C., De Zotti, M., Peggion, C., Biondi, B., Ballano, G., Formaggio, F., Toniolo,C. Chem. Biol. Drug Des. 75, 169-181 (2010).5. Formaggio, F., Broxterman, Q.B., Toniolo, C. In Goodman, M., Felix, A., Moroder, L., Toniolo, C.(Eds.) Houben-Weyl: Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics,Vol E22c, Thieme, Stuttgart, 2003, p. 292.383