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| C. Ciências Biológicas - 8. Genética - 2. Genética de Microorganismos |
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| CRYSTALLIZATION AND PRELIMINARY STRUCTURAL ANALYSIS OF THE Xanthomonas citri MALTOSE-BINDING PROTEIN (MalE) |
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| Cristiane Santos de Souza 1, Andrea Balan Fernandes 1, Carlos Henrique Inácio Ramos 2, Joao Alexandre Ribeiro Gonçalves Barbosa 2 e Luís Carlos de Souza Ferreira 1 |
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| 1- Laboratório de Biologia Molecular e Estrutural de Proteínas – Departamento de Microbiologia – Instituto de Ciências Biomédicas II – Universidade de São Paulo – São Paulo – Brazil
2- Laboratório Nacional de Luz Síncrotron, LNLS, Campinas - Brazil
, UNIVERSIDADE DE SÃO PAULO - USP |
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| Maltose uptake in gram-negative bacteria requires the presence of an ABC type transport system composed of a membrane-bound complex formed by two hydrophobic permease subunits (MalF and MalG) and two copies of the ATPase subunit (MalK). The last component, the maltose binding protein (MBP) or MalE, is the soluble periplasmic bindig component that specifically recognizes and brings the sugar to the membrane components where the uptake process takes place. In this work we report the purification and crystallization of plant pathogen Xanthomonas citri (Xac) MBP. The encoded protein is composed of 538 amino acids with a molecular weigth of 50,9 kDa and a pI of 5,89. Sequence alignment of Xac MPB amino acid sequence showed high similarity values when compared to orthologs found in other bacterial plant pathogens. The MPB was expressed in E. coli BL21 (DE3) as a soluble fusion protein with the His-tag at the N-terminal. The recombinant protein was purified by affinity chromatography with recovery yields of approximately 0,6 g/L under optimal inducing conditions. Circular dicroism (CD) revealed that the protein contained 38 percent alpha-helix and 15 percent beta-sheet structures. Samples of the recombinant protein, suspended at concentrations of 4 and 10 mg/ml in Tris 20 mM pH 8.0 and NaCl 50 mM, were submitted to crystallization trials with and without maltose (1:2). Bipyramidal crystals were obtained in the presence of maltose at 10 mg/ml with Hampton Research I (0.1M Na HEPES, pH 7.5; 0.8M Potassium Sodium Tartrate Tetrahydrate), Hampton Research II (0.1M MES pH 6.5, 12% PEG 20000 and 0.1M HEPES pH 7.5, 20% PEG 10000) and Jena Bioscences (0.1M Na HEPES pH 7.5, 0.2M Sodium acetate, 20% PEG 3000 and 25% PEG 2000 MME) kits. X-ray diffraction at 4 Å and 7Å were achieved and results are reported. Refinement of crystallographic analysis will contribute to the unraveling of MPB structure and a better understanding of its role on the physiology of it Xac.
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Instituição de fomento: Pró – Reitoria de Pesquisa USP; FAPESP.
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Trabalho de Iniciação Científica
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| Palavras-chave:
MalE; Xanthomonas citri; maltose |
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Anais da 57ª Reunião Anual da SBPC - Fortaleza, CE - Julho/2005
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