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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, 2010Conformational Studies of Toac-Analogues from New CytolyticPeptide Isolated from Hypsiboas AlbopunctatusEduardo F. Vicente 1 , Graziely F. Cespedes 1 , Edson Crusca Jr 1 ,Mariana S. Castro 2 , Maria José S. Mendes-Giannini 1 ,Luís Guilherme M. Basso 3 , Antonio J. Costa-Filho 3 ,Reinaldo Marchetto 1 , and Eduardo M. Cilli 11 UNESP – Univ Estadual Paulista, Araraquara, Brazil; 2 UnB – University of Brasília,Brasília, Brazil; 3 USP – Universidade de São Paulo, São Paulo, BrazilIntroductionAntibiotic resistant bacterial strains represent a global health problem with a strong socialand economic impact. Thus, there is an urgent need for the development of antibiotics withnovel mechanisms of action. Castro’s group isolated and determined from the skinsecretion of the frog Hypsiboas albopunctatus the new antimicrobial peptide called Hylin(hy - GWLDVAKKIGKAAFNVAKNFI/L). The sequence analysis of this peptide(http://ca.expasy.org/tools/blast/) revealed identity with ceratoxin – extracted from thefemale reproductive accessory glands of the dipteran insect Ceratitis capitata. The aim ofthe present work was to evaluate three analogues containing TOAC to supply informationabout the topology of this peptide in mimetic membrane and the pore formationmechanism. These analogues contain Ile in C-terminus and addition of TOAC (4-amino-4-carboxy-2,2,6,6-tetramethyl-piperidine-1-oxyl; [1,2]) in position 0 or replacing Trp 2 andAla 13 . These modifications allowed studying the conformational properties and itsinteraction with membrane models by EPR.Results and DiscussionThe peptides were synthesized manually according to the standard SPPS-N α -Fmoc(9-fluorenylmethyloxycarbonyl) protecting group strategy. The cleavage reaction wasperformed with anhydrous TFA (trifluoroacetic acid), TIS (triisopropylsilane) and waterPercentual dye released100806040200WTTOAC 0 -hyTOAC 2 -hyTOAC 13 -hy0 2 4 6 8 10Time (min)Fig. 1. Membrane permeabilizationof DPPE/DPPA/DPPC vesiclesinduced by 2 mmol L -1 of syntheticpeptides.(9:0.5:0.5). The crude spin-labeled peptides weresubmitted to alkaline treatment (pH 10, 4 h) forcomplete reversion (monitored by analytical HPLC)of the N-O protonation that occurs during the TFAreaction [3,4]. The peptides were purified bypreparative HPLC at pH 5.0. The synthesis andpurification of peptides by HPLC was efficient and ahigh purity level (≥ 96%) was obtained. Thebiological activity [5] of these peptides wasdetermined. All synthetic peptides exhibitedhemolytic, antimicrobial and antifungal activity. Thehemolytic activities of analogues TOAC 0 -hy (3 μmolL -1 ) and TOAC 13 -hy (1 μmol L -1 ) were bigger thanwild type (WT-hy) synthetic peptide (7 μmol L -1 ),except by TOAC 2 -hy (19 μmol L -1 ). Most peptidesshowed similar antimicrobial activity from WT-hy,except TOAC 0 -hy that showed high value of MIC(minimal inhibitory concentrations). To obtain moreinformation about the difference among the peptides permeabilization, studies in vesiclescontaining DPPC:DPPA:SM and DPPC:DPPA:DPPE (80:5:15; w:w:w) were performed(Figure 1). The data showed that TOAC 2 -hy has the lowest activity, followed by TOAC 0 -hy, TOAC 13 -hy and WT-hy. To understand the difference between the biological activities,the secondary structure was evaluated by CD spectroscopy in aqueous solution (at 25ºC andpH 7.0), presence of trifluoroethanol 60% (v/v) and vesicles. The CD studies demonstratedthat peptides in water had a random coil structure, except by TOAC 13 -hy, which had anα-helix structure. This is in accordance with the TOAC properties, which exhibits atendency to strongly promote helical conformations. In the presence of TFE or mimetic388