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60fluorescência fluorescence(u.a)4020NaCl 0,4MNaCl 4M00 5 10 15 20 25 30 35 40fractionsfraçãoFig. 2. CcdA41 affinity chromatography assays in columns containing immobilizedCcdBET2 ( ), CcdBET3 ( ) and CcdBCC1 ( ).The affinity chromatography studies revealed interactions among the CcdA41 and thepeptides fragments of CcdB. The interesting point was that the interactions were dependenton the Arg40-Leu50 sequence. This result prompted us to determine the binding parametersfor the systems mentioned above. The binding parameters were studied by following thequenching of the intrinsic fluorescence of the CcdA41 upon binding of the CcdBCC1 as hasalready described for CcdBET2 [4]. The plot of the relative CcdA41 fluorescence intensityat the emission wavelength of 350 nm ( ex = 280 nm) as a function of total CcdBCC1 wasperformed. The association constant (K a ) values to the CcdA41-CcdBCC1 were obtainedfrom the slope of the Stern-Volmer plot for identified static quenching [5], according toequation F 0 /F = 1 + K a [CcdBCC1]. The obtained values of 416 mmol/L -1 for K a are themeans of at least five measurements. The ability of the CcdB fragments to inhibit thesupercoiling reaction of DNA gyrase was investigated by titrating CcdB fragments into afixed concentration of enzyme and DNA. The minimum concentration that producedcomplete inhibition of supercoiling activity was termed the IC 100 [6]. In standardsupercoiling assays at 37°C with 3.4 nM of gyrase, a relaxed DNA (500 ng) substrate iscompletely negatively supercoiled in 1 h. CcdBET2 and CcdBET3 inhibited this reactionwith an IC 100 value of 15 and 120 µM, respectively. CcdBCC1 did not inhibit the DNAgyrase. This finding supports the theory that the three terminal residues (WGI) of CcdBplays a crucial role in DNA gyrase-CcdB complex formation [2].AcknowledgmentsThis work was supported by FAPESP, in the form of research assistance (03/04492-3) and scholarship(IC 07/08052-9 - Cotrim C.Ap.) and CNPq in the form of PhD scholarships (Garrido S.S. and TrovattiE.) and research (Marchetto R.).References1. Couturier, M., Bahassi, E.M., Van Melderen, L. Trends in Microbiology 6, 269 (1998).2. Loris, R., Dao-Thi, M., Bahassi, EM.,Van Melderen, L., Poortmans, F., Liddington, R., Couturier,M., Wyns, L. J. Mol. Biol. 285, 1667 (1999).3. Marchetto, R., Nicolás, E., Castillo, N., Bacardit, J., Navia, M., Vila, J., Giralt, E. J. Pep. Sci. 7, 27(2001).4. Cotrim, C.A., Garrido, S.S., Trovatti, E., Marchetto, R. Quim. Nova. 33, 4, 841, (2010).5. Garrido, S.S., Scatigno, A., Trovatti, E., Marchetto, R. J. Pep. Res. 65, 5, 502, (2005).6. Trovatti, E., Cotrim, C. Ap., Garrido, S.S., Barros, R., Marchetto, R. Bioorg. Med. Chem. Lett. 18,6161-6164 (2008).567