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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, 2010Comparison of the Mechanism of Action of AntimicrobialPeptides on Giant Unilamellar Vesicles via Optical MicroscopyMarta N.C. Martins, Tatiana M. Domingues, Karin A. Riske, andAntonio MirandaDepartment of Biophysics, Federal University of São Paulo, São Paulo, SP,04044-020, BrazilIntroductionAntimicrobial peptides (AMPs) are important components of the innate defense system ofplants and animals against microorganisms, such as bacteria and fungi. Most AMPs arecationic and amphipathic, features which are essential for their interaction with the lipidphase. Initially, the cationic aspect of AMPs ensures an accumulation at the membranesurface of the microorganisms, rich in negatively charged lipids. Subsequently, theamphiphilic character of the AMPs facilitates their insertion into lipid bilayers, withformation of pores and/or disruption of membranes. Due to its broad antibacterial andantifungal activity spectrum AMPs are a potential target for the development of alternativetherapeutic applications. In order to understand the mechanism of lytic action we usedoptical microscopy to compare the interaction of different AMPs (Table 1) with giantunilamellar vesicles (GUVs) composed of mixtures of the neutral lipid palmitoyl-oleoylphosphatidylcholine (POPC) with the negatively charged lipid palmitoyl-oleoylphosphatidyl-glycerol(POPG) (1:1) so as to mimic bacterial cell membrane.Results and DiscussionPeptides were synthesized by SPPS, purified by preparative RP-HPLC and characterized byAAA and LC-MS [1]. GUVs were grown by the electroformation method [2,3] in 0.2 Msucrose and later diluted into 0.2M glucose. This created a sugar asymmetry whichenhanced the optical contrast with phase contrast microscopy. The GUVs were then placedin an observation chamber. A solution of peptide was injected and a population of vesicleswas followed with time in order to evaluate the lytic activity of the different AMPs studied(Table 1). Our results (Figure 1) indicated that gomesin [4], polyphemusin II [5] andtachyplesin I [6] induced sudden burst of GUVs, with accompanying fast release of theentrapped volume. Stable pores, which result in leaky vesicles, were not observed. On theother hand, magainin II [7] and protegrin I [8] were able to induce the opening of stablepores through which the initial sucrose/glucose asymmetry of the GUVs was lost.Table 1. Antimicrobial peptides employed in this study.Namegomesinpolyphemusin IItachyplesin ISequence aZ-C-R-R-L-C-Y-K-Q-R-C-V-T-Y-C-R-G-R-NH 2R-R-W-C-F-R-V-C-Y-K-G-F-C-Y-R-K-C-R-NH 2K-W-C-F-R-V-C-Y-R-G-I-C-Y-R-R-C-R-NH 2magainin II G-I-G-K-F-L-H-S-A-K-K-F-G-K-A-F-V-G-E-I-M-N-S-NH 2protegrin I R-G-G-R-L-C-Y-C-R-R-R-F-C-V-C-V-G-R-NH 2a Z = pyroglutamic acid404