Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23985
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dc.contributor張邦彥zh_TW
dc.contributorPang-Yen Changen_US
dc.contributor蔡榮宗zh_TW
dc.contributorRong-Tzong Tsaien_US
dc.contributor.advisor胡念台zh_TW
dc.contributor.advisorNien-Tai Huen_US
dc.contributor.author王琮博zh_TW
dc.contributor.authorWang, Tsung-Poen_US
dc.contributor.other中興大學zh_TW
dc.date2009zh_TW
dc.date.accessioned2014-06-06T07:21:43Z-
dc.date.available2014-06-06T07:21:43Z-
dc.identifierU0005-1208200823013400zh_TW
dc.identifier.citation李憲明 (2001) 第二類型蛋白分泌機制中內膜蛋白XpsN與其它Xps蛋白之交互作用 中興大學 農業生物科技學研究所 游婷婷 (2003) 十字花科黑腐病菌中XpsF與XpsL或XpsE蛋白交互作用之分析 碩士論文 中興大學 生物科技研學究所 李孟訓 (2004) 十字花科黑腐病菌第二型分泌系統中類纖毛結構的主要組成蛋白XpsG與非類纖毛蛋白間相互作用關係之研究 博士論文 中興大學 生物化學研究所 范芝瑋 (2007) XpsF位於細胞內的區域與XpsLN及與XpsE蛋白之相互作用關係 碩士論文 中興大學 生物化學研究所 趙怡姍 (2007) XpsE蛋白N2區域在XpsE形成多聚體及其與XpsL蛋白相互作用中的重要性 碩士論文 中興大學 生物化學研究所 Abendroth, J., Murphy, P., Sandkvist, M., Bagdasarian, M. and Hol, W.G.J. (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 348:845-855 Camberg, J.L. and Sandkvist, M. (2005) Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacterial 187.1:249–256 Camberg, J.L., Johnson, T.L., Patrick, M., Abendroth, J., Hol, W.G.J. and Sandkvist, M. (2006) Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. EMBO J 26:19-27 Chen, L.Y., Chen, D.Y., Miaw, J. and Hu, N.T. (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J Biol Chem 271:2703-2708 Chen, Y., Shiue, S.J., Huang, C.W., Chang, J.L., Chien, Y.L., Hu, N.T. and Chan, N.L. (2005) Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem 280:42356-42323 Durand, E., Michel, G., Voulhoux, R., Kürner, J., Bernadac, A. and Filloux, A. (2005) XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J Biol Chem 280:31378-31389 Hu, N.T., Lee, P.F. and Chen, C.C. (1995) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. Mol Microbiol 18:769-777 Hu, N.T., Leu, W.M., Lee, M.S., Chen, A., Chen, S.C., Song, Y.L. and Chen, L.Y. (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 Korotkov, K.V. and Hol, W.G.J. (2008) Structure of the GspK–GspI–GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nat Struct Mol Biol 15:462-468 Lee, H.M., Tyan, S.W., Leu, W.M., Chen, L.Y., Chen, D.C. and Hu, N.T. (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 Lee, M.S., Chen, L.Y., Leu, W.M., Shiau, R.J. and Hu, N.T. (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 Lindeberg, M., Salmond, G.P.C. and Collmer, A. (1996) Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mol Microbiol 20:175-190 Nunn, D.N. and Lory, S. (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W. J Bacteriol 175:4375-4382 Planet, P.J., Kachlany, S.C., DeSalle, R. and Figurski, D.H. (2001) Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci USA 98:2503-2508 Rial, D.V. and Ceccarelli, E.A. (2002) Removal of DnaK contamination during fusion protein purifications. Protein Expr Purif 25:503-507 Robien, M.A., Krumm, B.E., Sandkvist, M. and Hol, W.G.J. (2003) Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae. J Mol Biol 333:567-674 Satyshur, K.A., Worzalla, G.A., Meyer, L.S., Heiniger, E.K., Aukema, K.G., Misic, A.M. and Forest, K.T. (2007) Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure 15:363-376 Sandkvist, M., Bagdasarian, M., Howard, S.P. and DiRita, V.J. (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 Sandkvist, M. (2001) Biology of type II secretion. Mol Microbiol 40: 271-283 Sauvonnet, N., Vignon, G., Pugsley, A.P. and Gounon, P. (2000) Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J 19:2221-2228 Savvides, S.N., Yeo, H.J., Beck, M.R., Blaesing, F., Lurz, R., Lanka, E., Buhrdorf, R., Fischer, W., Haas, R. and Waksman, G. (2003) VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. EMBO J 22:1969-1980 Shiue, S.J., Kao, K.M., Leu, W.M., Chen, L.Y., Chan, N.L. and Hu, N.T. (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J 25:1426-1435 Shiue, S.J., Chien, I.L., Chan, N.L., Leu, W.M. and Hu, N.T. (2007) Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Mol Microbiol 65:401–412 Tsai, R.T., Leu, W.M., Chen, L.Y. and Hu, N.T. (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. Biochem J 367:865-871 Vorhöltera, F.J., Goesmannc, S.S.A., Bekelc, L.K.T., Kaiserd, O., Linkec, B., Patschkowskid, T., Rückertd, C., Schmide, J., Sidhub, V.K., Sieberf, V., Tauchd, A., Wattb S.A., Weisshaard B., Beckerd, A., Niehausb, K. and Pühler, A. (2008) The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis. J Biotechnol 134:33-45 Yamagata, A. and Tainer, J.A. (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. EMBO J 26: 878-890zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/23985-
dc.description.abstract第二型分泌系統 (T2SS) 是十字花科黑腐病菌分泌水解酵素、攻擊寄主的主要機制,此機制也出現在許多不同的致病菌株中,感染動、植物並造成疾病。 在T2SS中,位於cytosol的XpsE是唯一具ATP結合及水解能力的組成蛋白,推測為T2SS運轉提供所須要的能量。 先前研究指出,XpsE ATP水解活性可能受到其N1區域的抑制,而內膜蛋白XpsL可透過與XpsE N1區域結合,移去N1的抑制而達到刺激XpsE ATP水解活性的效果。 XpsE的N2區域位於N1區域及其C區域ATP水解活性中心之間,推測N2區域可能居中參與XpsE蛋白構形變化之訊息傳遞以及ATP水解活性之調控。 XpsE本身可聚合成多聚體,可能以六聚體的形式推動T2SS的運轉。 XpsE同源蛋白的立體結構暗示,N2區域亦可能參與六聚體中分子間的結合。 本研究選擇高保留性的D202以alanine定點突變的方式進行分析。 結構顯示XpsE同源蛋白N2區域的D202同源胺基酸與C區域所結合的ATP靠近。 D202A突變蛋白失去正常in vivo分泌功能,並具有干擾正常XpsE行使功能的特性。 本研究希望藉由D202A突變蛋白和正常XpsE蛋白生化特性的比較,以瞭解胺基酸D202在XpsE行使功能時所扮演的角色。 結果發現D202A突變蛋白的許多特性都與正常XpsE無明顯差異,它能正常結合ATP,沒有喪失形成多聚體的能力,且仍然可以與XpsL結合並受到XpsL刺激ATP水解活性。 但是,D202A突變蛋白單獨的ATP水解活性略低,同時對ADP可能有異常的結合能力。 最後以四個已知XpsE同源蛋白的結構為依據,分析N2區域以及D202保留性胺基酸鄰近的構造,推測胺基酸D202可能主要參與XpsE水解ATP的過程或其後的下游步驟。zh_TW
dc.description.abstractT2SS is the mechanism required for Xanthomonas campestris pv. campestris to secret hydrolytic enzymes in the invasion of its host. Widespread in pathogenic bacteria, the secretion system leads to animal and plant diseases. XpsE, a cytosolic protein, is the only component in T2SS that possesses ATP binding and hydrolyzing activities and may work as the molecular motor. Previous studies indicated that the N1 domain of XpsE is inhibitory to its ATPase activity exihibited by its C domain. An inner membrane protein XpsL acts as a positive regulator of the XpsE ATPase, presumably by binding to the XpsE N1 domain and alliviating its inhibition effect. An N2 domain physically located between N1 and C domain of XpsE may coordinate conformational change related ATPase activity regulation. XpsE forms homo-multimer and may function as hexamer in T2SS. Crystal structures of XpsE homologues suggest that the N2 domain may be involved in inter subunit association in hexamer. This study is focused on the analysis of an XpsE mutant that is mutated at the conserved residue D202 in the N2 domain. The XpsE(D202A) is no longer functional in secretion. It also interferes with normal secretion when introduced into the wildtype strain. In crystal structures of XpsE homologue, the D202 equivalent residues are close to ATP bound to the C domain. To get better understanding of functional role of the residue D202 in XpsE, ATP binding and ATP hydrolyzing activities of the mutant were analyzed in parellel with the wildtype XpsE. In most assays, no significant alteration was observed. The mutant remains capable of ATP binding, appears to exihibit normal oligomerization activity and XpsLN binding. However, it exihibits slightly lower intrinsic ATPase activity and deviated ADP binding. By inspecting surroundings of D202 equivalents in the solved crystal structures, we speculated that the D202 residue of XpsE may be required for steps downstream of ATP hydrolysis by XpsE.en_US
dc.description.tableofcontents摘要 i Abstract ii 目次 iii 前言 1 實驗方法 8 一、螢光測試 8 (一) 蛋白的表現、純化及處理 8 (二) 利用螢光測試XpsE與TNP-ATP的結合能力 9 二、ATP水解活性的測試 10 (一) 蛋白的表現、純化及處理 10 (二) ATP水解活性測試及分析 11 (三) ATP水解活性的計算方式 12 (四) MBP-XpsLN的表現與純化 13 (五) ADP競爭ATP水解活性測試 13 結果 14 一、D202A突變對XpsE結合ATP的能力無顯著影響 14 二、D202A突變造成XpsE的ATP水解活性略為下降 15 三、D202A突變不影響XpsE的ATP水解活性隨蛋白濃度提高而上升的現象 15 四、XpsE(D202A) 突變蛋白的 ATP水解活性仍受到XpsLN的刺激 16 五、ADP抑制ATP水解活性實驗暗示D202A定點突變蛋白較XpsEwt可能對ADP有較強的結合能力 17 討論 18 D202位於N2區域與相鄰分子C區域的接觸介面 18 D202可能與分子內N2區域的R286形成salt bridge 19 D202在N1、N2區域間訊息傳遞中可能扮演的角色 20 參考文獻 22 圖一 27 圖二 28 圖三 29 圖四 31 圖五 32 圖六 33 圖七 36 表一、菌種 37 表二、表現質體 37 表三、 XpsEwt、XpsE(D202A) 蛋白單獨或添加MBP-XpsLN時的ATP水解活性 39 附錄 40zh_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=U0005-1208200823013400en_US
dc.subjecttype II secretion systemen_US
dc.subject第二型分泌機制zh_TW
dc.subjectATPaseen_US
dc.subject水解活性zh_TW
dc.titleXpsE N2區域的重要性:XpsE(D202A) ATP 水解活性及ATP、ADP結合能力的分析zh_TW
dc.titleSignificance of the XpsE N2 domain: Analysis of the XpsE(D202A) ATPase activity and its ATP, ADP bindingen_US
dc.typeThesis and Dissertationzh_TW
item.cerifentitytypePublications-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.grantfulltextnone-
item.openairetypeThesis and Dissertation-
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