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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, 2010Membrane Insertion of para-Cyanophenylalanine LabeledAlamethicin Analogues. Correlation of Fluorescence andInfrared Absorption DataSara Bobone 1 , Marta De Zotti 2 , Annalisa Bortolotti 1 , Gema Ballano 2 ,Fernando Formaggio 2 , Claudio Toniolo 2 , and Lorenzo Stella 11 Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata",Rome, 00133, Italy; 2 ICB, Padova Unit, CNR, Department of Chemistry,University of Padova, Padova, 35131, ItalyIntroductionDifferent classes of peptides, such as antimicrobial, cell penetrating, and fusogenicpeptides, exert their bioactivities by interacting with cellular membranes. Therefore, thedetermination of their position and orientation inside a lipid bilayer is a fundamental step inthe characterization of their mechanism of action. In this respect, the α-amino acid paracyanophenylalanine(pCNPhe) is a very promising probe, since it can be employed both influorescence and in IR absorption experiments [1,2]. Its fluorescence quantum yield issignificant (0.11, as compared to 0.025 for Phe), and the C≡N stretching vibrationaltransition is located around 2230 cm -1 (i.e., far from the water background absorption) andis sensitive to the medium polarity.Results and DiscussionIn this study, we exploited the peculiar properties of pCNPhe to investigate the membraneinteraction of the [Glu(OMe) 7,18,19 ] alamethicin analogue by synthesizing three monolabeledpeptides in which Ala 4 (AL4), Val 9 (AL9) or Val 15 (AL15) are substituted bypCNPhe. Liposome leakage kinetics data indicated that the label does not perturbsignificantly the peptide activity. Furthermore, the pCNPhe fluorescence (Figure 1) issensitive to the fluorophore environment, allowing a characterization of peptideaggregation and water-membrane partition. These experiments showed a very similarbehavior for all analogues.Normalized fluorescence [a.u.]10.50Al4Al9Al15290 300 310 320 330 340 350λ[nm]Fig. 1. Fluorescence spectra of the three alamethicin analogues AL4, AL9 and AL15 inphosphate buffer, 140 mM NaCl, 0.1 mM EDTA, pH=7.4., exhibiting the characteristicemission bands of the pCNPhe chromophore.372