Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23974
DC FieldValueLanguage
dc.contributor陳凌雲zh_TW
dc.contributor李憲明zh_TW
dc.contributor呂維茗zh_TW
dc.contributor詹迺立zh_TW
dc.contributor.advisor胡念台zh_TW
dc.contributor.author陳歆怡zh_TW
dc.contributor.authorChen, Hsin-Yien_US
dc.contributor.other中興大學zh_TW
dc.date2008zh_TW
dc.date.accessioned2014-06-06T07:21:41Z-
dc.date.available2014-06-06T07:21:41Z-
dc.identifierU0005-3107200711213400zh_TW
dc.identifier.citation參考文獻 1. Abendroth J, Murphy P, Sandkvist M, Bagdasarian M, Hol WG (2005) The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. J Mol Biol 348: 845-55 2. Bitter W, Koster M, Latijnhouwers M, de Cock H, Tommassen J (1998) Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27: 209-19 3. Chien IL (2002) Purification and characterization of the XpsE protein of the type II secretion apparatus of Xanthomonas campestris pv. campestris. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 4. Camberg JL, Sandkvist M (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol 187: 249-56 5. Chen LY, Chen DY, Miaw J, Hu NT (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem 271: 2703-8 6. Chen Y, Shiue SJ, Huang CW, Chang JL, Chien YL, Hu NT, Chan NL (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem 280: 42356-63 7. Dums F, Dow JM, Daniels MJ (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol Gen Genet 229: 357-64 8. Filloux A (2004) The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694: 163–179 9. Genin S, Boucher, CA (1994) A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mol Gen Genet 243: 112-118 10. Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 174: 2679–2687 11. Hu NT, Hung MN, Liao CT, Lin MH (1995) Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology 141: 1395-406 12. Hu NT, Leu WM, Lee MS, Chen A, Chen SC, Song YL, Chen LY (2002) XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes. Biochem J 365: 205–211 13. Kao KM (2005) Type II secretion apparatus of Xanthomonas campestris: Determination of ATPase activity of XpsE and mutant analysis of XpsE with mutations at nucleotide-binding motifs. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 14. Lee HM, Tyan SW, Leu WM, Chen LY, Chen DC, Hu NT (2001) Involvement of the XpsN protein in formation of the XpsL-XpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. J Bacteriol 183: 528–535 15. Lee MS, Chen LY, Leu WM, Shiau RJ, Hu NT (2005) Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. J Biol Chem 280: 4585–4591 16. Linderoth NA, Simon MN, Russel M (1997) The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 278: 1635-8 17. Nouwen N, Ranson N, Saibil H, Wolpensinger B, Engel A, Ghazi A, Pugsley AP (1999) Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc Natl Acad Sci U S A 96: 8173-7. 18. Nunn DN, Lory S (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 75: 4375-82 19. Py B, Loiseau L, Barras F (1999) Assembly of the type II secretion machinery of Erwinia chrysanthemi: Direct interaction and associated conformational change between OutE, the putative ATP binding component and the membrane protein OutL. J Mol Biol 289: 659–670 20. Robien MA, Krumm BE, Sandkvist M, Hol WG (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333: 657–674 21. Sandkvist M (2001) Biology of type II secretion. Mol Microbiol 40: 271-83 22. Sandkvist M, Bagdasarian M, Howard SP, DiRita VJ (1995) Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J 14: 1664–1673 23. Sandkvist M, Keith JM, Bagdasarian M, Howard SP (2000) Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae. J Bacteriol 182: 742-8 24. Shiue SJ, Kao KM, Leu WM, Chen LY, Chan NL, Hu NT (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25: 1426-35 25. Shiue SJ, Chien IL, Chan NL, Leu WM, Hu NT (2007) Mutation of a key residure in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol Microbiol 65: 401-412 26. Thomas JD, Reeves PJ, Salmond GP (1997) The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF. Microbiology 143: 713-20 27. Tsai RT, Leu WM, Chen LY, Hu NT (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J 367: 865-71 28. Tseng YN (2006) Significance of the N-terminal domain of XpsE in its interaction with XpsL analyzed by site-directed mutation. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 29. Wei CJ (2006) Analysis of conformation change at the N domain of XpsE protein by utilizing fluorescence probes. Master thesis. Graduate Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 30. Yo TT (2003) Detection of interactions between XpsF and XpsL, or XpsE, in the type II secretion apparatus of Xanthomonas campestris pv. Campestris. Master thesis. Graduate Institute of Agricultural Biotechnology, National Chung-Hsing University, Taichung, Taiwan, R. O. C. 31. Yamagata A, Tainer JA (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J 26: 878-90zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/23974-
dc.description.abstract十字花科黑腐病菌中的第二型蛋白分泌系統由12個蛋白所組成,能夠將在胞內合成的多種水解酶分泌至胞外,破壞植物細胞的表面構造。XpsE是第二型蛋白分泌系統中唯一的胞內蛋白,具有ATPase活性,在與ATP結合後,會聚合成六倍體且與內膜上的XpsL的N端 (XpsLN) 交互作用,XpsE主要是以N端區域與XpsLN結合。根據XpsE的N端晶體結構得知,XpsEN有open form與closed form兩種構形,推測只存在於closed form構形中的疏水性表面區域,可能參與XpsE與XpsLN的結合。先前藉由三定點突變 (V11A,L25A,L39A) 的實驗發現突變蛋白XpsE(TM11,25,39) 明顯降低與XpsLN結合的能力,並喪失分泌功能。本研究為進一步了解此三定點突變蛋白與正常XpsE的差異,進行三方面的分析。首先,利用疏水性螢光探針ANS對XpsE與XpsE(TM11,25,39) 分別進行疏水性結合的標定,發現XpsE(TM11,25,39) 的螢光訊號明顯較XpsE強,暗示此三定點突變可能導致XpsE結構產生變化,暴露出較多的疏水性區域。其次,利用gel filtration比較由affinity column純化得到的XpsE與 XpsE(TM11,25,39) 其monomer與oligomer的分佈情形,發現 XpsE(TM11,25,39) 相對於XpsE有較高比例的oligomer存在。最後,在ATPase活性方面,發現在高鹽 ( 500 mM NaCl ) 以及含大量ATP的緩衝液清洗的純化方式下,測得XpsE(TM11,25,39) 蛋白的ATPase活性約為野生型XpsE蛋白的四倍。zh_TW
dc.description.abstractType II secretion apparatus of Xanthomonas campestris pv. campestris is constituted of twelve proteins, and mediates the translocation of hydrolytic enzymes from periplasm to the milieu, possibly to destroy the surface component of plant cell. XpsE is the only cytoplasmic protein component in the type II secretion apparatus, and exhibits ATPase activity. ATP binding triggers XpsE to form hexamer as well as its association with the N-terminal domain of the cytoplasmic membrane protein XpsL (XpsLN). XpsE utilizes its N-terminal domain to associate with XpsLN. Two crystal structures of XpsEN were revealed, designated as an open and a closed form that differ at their N-termini. A hydrophobic patch detected only in the closed form was proposed to be involved in the interaction between XpsE and XpsLN. Triple alanine mutations at Val11, Leu25, Leu39 caused XpsE lose secretion function and the ability to bind XpsL. To further characterize the mutant XpsE(TM11, 25, 39), it was analyzed in parallel with wide type XpsE in three aspects. First of all, a fluorogenic probes ANS was utilized for monitoring possible difference in their hydrophobicity. The fluorescence signal of XpsE(TM11, 25, 39) is significantly stronger than that of XpsE, suggesting conformation change in XpsE conformation may have been caused by triple mutation. Secondly, size exclusion chromatography of the affinity column-purified Strep-tagged XpsE and XpsE(TM11, 25, 39) revealed difference in the proportion of their monomer and oligomer. The XpsE(TM11, 25, 39) appeared as oligomeric form at higher proportion than the wild type XpsE. Finally, the ATP activity of the XpsE(TM11, 25, 39) that has been washed with buffer containing high salt and large amount of ATP is about four-times that of the wide-type XpsE.en_US
dc.description.tableofcontents前言………………………………………………………………………1 材料與方法………………………………………………………………6 一、材料…………………………………………………………………6 1.菌種與質體……………………………………………………………6 2.引子....................................................6 3.培養基、試劑與緩衝溶液……………………………………………6 二、實驗方法……………………………………………………………6 1.小量質體DNA之萃取.......................................6 2.勝任細胞之製備………………………………………………………7 3.轉形....................................................7 4.菌種甘油保存法………………………………………………………7 5.XpsE蛋白及XpsE突變蛋白的大量表現………………………………7 6.XpsE(TM11, 25, 39) 蛋白的大量表現.......................8 7.一般蛋白純化............................................8 8.蛋白質SDS-PAGE電泳分析..................................8 9.西方墨點法..............................................8 10.化學冷光法.............................................9 11.一般呈色法.............................................9 12.Coomassie blue染色估量法...............................9 13.ANS螢光測試............................................9 14.分子篩管柱層析........................................10 15.陰離子交換樹脂色層分析…………………………………………10 16.野生型XpsE蛋白之ATPase活性測定………………………………10 A.蛋白純化-ATP與DDM沖洗.................................10 B.ATPase測定.............................................11 17.XpsE(TM11, 25, 39)蛋白之ATPase活性測定................11 A.蛋白純化...............................................11 a.ATP沖洗-低鹽 (200 mM NaCl)……………………………………11 b.ATP沖洗-高鹽 (500 mM NaCl)……………………………………11 c.ATP與DDM沖洗...........................................12 B.ATPase測定.............................................12 結果.....................................................13 一、V11A, L25A, L39A三點突變導致XpsE蛋白的三級結構呈現顯著變化…13 二、V11A, L25A, L39A三點突變造成XpsE蛋白較野生型蛋白易形成多聚體…14 三、XpsE(TM11, 25, 39) 蛋白的ATPase活性分析…………………15 四、XpsE蛋白單體與陰離子交換樹脂的結合力較多聚體弱.......20 討論.....................................................21 參考文獻.................................................25 圖.......................................................29 表.......................................................46 附錄.....................................................50 附圖.....................................................55zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3107200711213400en_US
dc.subjectXpsE proteinen_US
dc.subjectXpsE蛋白zh_TW
dc.titleXpsE蛋白N端突變對其結構與功能之影響zh_TW
dc.titleInfluence of mutations at the N domain of XpsE on its structural and functional propertiesen_US
dc.typeThesis and Dissertationzh_TW
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
Appears in Collections:生物化學研究所
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