| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 98 === XpsE is the only cytoplasmic protein component of the Xanthomonas campestris pv. campestris type II secretion system. It can be divided into N and C domain, and its C domain contains four conserved nucleotide binding motifs, which exhibit ATPase activity. As suggested from previous in vitro studies, ATP binding to XpsE triggers its hexamerization, and XpsE plays an important part in supplying energy for secretion process by hydrolyzing ATP. In the hexameric structure of aaPilT, an XpsE homologues, three interfaces between two neighboring subunits were revealed. The interface between N2 and C1 covers the largest area and contains most highly conserved residues. It was suggested that the interactive residues located at the N2-C1 interface may be important for protein hexamerization. Sequence alignment indicates that R385 and D387 may be located at the interactive interface of two neighboring XpsE. In this study, we conducted biochemical analysis of the mutant XpsE(R385A) and XpsE(D387A) in parallel with the wild type XpsE to get better understanding of the functional role of R385 and D387 in XpsE. By performing size exclusion chromatography and Western blot, we observed reduction in hexamer content of the mutant XpsE(R385A) and XpsE(D387A) relative to that of the wild type XpsE. Their ATP hydrolysis activity was reduced by 10 fold. In order to minimize possible contaminating ATPase and to raise protein homogeneity, we purified the proteins by performing affinity chromatography followed by anion exchange chromatography, from which homogeneous monomers with little contaminating ATPase were obtained. Following concentration, the monomeric protein was analyzed for ATPase activity by using a non-radioisotopic ATPase assay developed in this study. In summary, the experimental evidences presented in this study suggest mutation of the highly conserved residues possibly located at interactive interface of neighboring XpsE, R385 and D387, to alanine diminishes the ability of XpsE in hexamerization and reduces its ATP hydrolysis activity.
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