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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, 2010CcdB Toxin Peptides Derivatives and its Interactions with anAnalogue of Bacterial CcdA AntitoxinSaulo S. Garrido, Camila Ap. Cotrim, Davi B. Delfino,Anderson Garcia, Luiz C.B. Barbosa, and Reinaldo MarchettoInstitute of Chemistry, UNESP – Univ Estadual Paulista, Dept. Biochemistry andChemistry Technology, Araraquara – São Paulo, BrazilIntroductionDNA gyrase is an essential enzyme for maintaining the topological state of DNA inbacterial cells, making it a potential target for various antibacterial agents, including CcdBtoxin [1]. CcdB is a protein of 11.7 kDa, which together with its antidote CcdA, constitute aprogrammed cell death system in bacteria. Cell death is prevented when the antidote ispresent, which forms a stable complex with CcdB toxin. The design and synthesis ofpeptide analogues of CcdB, structurally modified to prevent the formation of CcdA-CcdBcomplex could result in a new family of peptide DNA gyrase inhibitors. In this context, wehave synthesized by solid phase methodology, a series of linear peptides derived fromCcdB protein, and studied if the loop formed by Arg40-Leu50 sequence is really importantin the process of interaction with the CcdA.Results and DiscussionAs an approach to understanding the CcdA-CcdB interactions, CcdA41, a polypeptidederived from CcdA, was synthesized and its interaction with CcdB peptide fragmentsanalyzed. The CcdB peptide fragments (Figure 1) were built based on crystal structure ofthe natural CcdB including the C-terminal -helix, the loop Arg40-Leu50 of the wingsheet, the N-terminal -sheet (Met1 to Leu9) and the residues Gly100 and Ile101 that seemto play a key role in the formation of CcdB-GyrA complex [2]. All peptides weresynthesized by solid phase methodology employing Fmoc/tBu strategy and were analyzedand purified by high performance liquid chromatography (HPLC) with final yield in therange of 20% for all sequences. The chemical identity was confirmed by mass spectrometry(positive ES-MS): 4942, 4872, 3459 and 2633 g/mol (CcdA41, CcdBET2, CcdBET3 andCcdBCC1 respectively).32 MQNEARRLRAERWKAENQEGMAEVARFIEMNGSFADENRDW 72 CcdA411 MQFKVYTYK 9 -Z- 40 RLLSDKVSREL 50 -Z- 84 SHRENDIKNAINKMFWGI 101 CcdBET21 MQFKVYTYK 9 -Z- 84 SHRENDIKNAINKMFWGI 101 CcdBET334 IPLASARLLSDKVSRELYPVVHIG 57 CcdBCC1Fig. 1. Primary structure of CcdA41 and synthetic peptides based on CcdB toxin.The peptides immobilized on Sepharose resin were used as supports for affinitychromatography studies, a way to evaluate the CcdA-CcdB interactions [3]. Thechromatographic behaviors of CcdA41 using the three columns containing immobilizedCcdBET2, CcdBET3 and CcdBCC1, respectively are shown in Figure 2. CcdA41 hadaffinity for the columns of immobilized CcdBET2 and CcdBCC1 and was eluted byaddition of 0.4 mol.L -1 NaCl in Tris buffer (10 mol.L -1 Tris.HCl / 20 mmol.L -1 NaCl / 5mmol.L -1 MgCl 2 , pH 7.2). When the Arg40-Leu50 sequence was absent (column ofimmobilized CcdBET3), the CcdA41 had no affinity to the column.566