| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 94 === Type II secretion apparatus is utilized by Xanthomonas campestris pv. campestris for secreting hydrolytic enzymes to infect plants on their leaves. XpsE, without any membrane spanning sequence, is the only cytoplasmic protein in type II secretion apparatus. It consists of four conserved nucleotide binding motifs, and is postulated to provide energy for the secretion process. A truncated XpsE mutant devoid of the N-terminal 36 residues (XpsE∆36) greatly affects its association with the inner membrane protein XpsL. To further understand significance of the N-terminal region of XpsE in its interaction with XpsL, I performed alanine mutation of chosen hydrophobic residues at the N-terminal region of XpsE and looked for mutations that cause XpsE nonfunctional in secretion. In this study, I observed that single mutations V11A, L15A or L25A in XpsE did not affect its normal function in secretion. Of all double mutants constructed, only the mutations at Val11 and Leu25 affect secretion slightly, with 54% secretion ability remaining. Similar level of secretion was also observed for one of the triple mutations V11A, L15A, L25A. In contrast, the other triple mutations V11A, L25A, L39A significantly affect the secretion function of XpsE, reduced to a level of 13% that of the wild-type XpsE, suggesting that intactness of the three hydrophobic residues Val11, Leu25 and Leu39 all together are required for normal function of XpsE. Triple alanine mutations at these three residues of XpsE did not affect its protein abundance. Analysis of circular dichroism and tryptophan fluorescence spectroscopy indicated that the secondary and the tertiary structure of XpsE were probably not significantly changed by the triple alanine mutations. In vitro pull down assay revealed that triple alanine mutations at Val11, Leu25 and Leu39 caused XpsE lose the ability to bind XpsL. This result implies that the hydrophobic patch constituted of the residues Val11, Leu25 and Leu39 are necessary for the association of XpsE with XpsL, which in turn is required for XpsE to be functional.
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