Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Revealing the Lytic Mechanism of the Antimicrobial PeptideGomesin by Optical Microscopy of Giant Unilamellar Vesiclesand Isothermal Titration CalorimetryTatiana M. Domingues 1 , Joachim Seelig 2 , Karin A. Riske 1 , andAntonio Miranda 11 Department of Biophysics, Federal University of São Paulo, São Paulo, 04044-020,Brazil; 2 Department of Biophysical Chemistry, Biocenter of the University of Basel,4051, Basel, SwitzerlandIntroductionGomesin (Gm) is a potent cationic antimicrobial peptide from a Brazilian spider [1,2]. Herewe use optical and fluorescence microscopy to study the interaction of Gm, its low activelinear analogue, [Ser 2,6,11,15 ]-Gm (GmL), and a fluorescent labeled analogue, Gm-Rh, withgiant unilamellar vesicles (GUVs, 10 µm) composed of mixtures of the neutral lipid POPCwith the negatively charged lipid POPG or cholesterol, so as to mimic bacterial andmammalian cell membranes, respectively. We observed the effect of injecting a peptidesolution with a micropipette close to GUVs. As a result of peptide-lipid interaction, GUVsburst suddenly. Stable pores, which result in leaky vesicles, were not observed. These factslead us to conclude that Gm and GmL disrupt the membrane via the carpet model [3]. GmLexhibited lower lytic activity as compared to Gm, but this difference vanished at highPOPG molar fraction. Additionally, the interaction of Gm and GmL with large unilamellarvesicles (LUVs, 100 nm) of various POPC:POPG ratios was investigated with isothermaltitration calorimetry (ITC). Binding of GmL to negatively charged vesicles is anexothermic process for all POPC:POPG ratios investigated. On the other hand, the bindingof Gm entails an exothermic and an endothermic component; the latter is more pronouncedat low POPG ratio and vanishes for 50 mol% POPG.Results and DiscussionDifferent experimental setups were used to study the interaction between the antimicrobialpeptides and the vesicles (GUVs and LUVs). First we observed the effect of injectingFig. 1. Heat flow vs. time measured with ITC. The syringe was loaded with 6 mM lipid andthe cell was filled with 10 µM peptide in all experiments. T = 25 o C, 10 mM Phosphatebuffer, pH 7.4400