Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23826
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dc.contributor.advisor周三和zh_TW
dc.contributor.author蔡穎德zh_TW
dc.contributor.authorTsai, Ying-Deren_US
dc.contributor.other中興大學zh_TW
dc.date2006zh_TW
dc.date.accessioned2014-06-06T07:21:14Z-
dc.date.available2014-06-06T07:21:14Z-
dc.identifier.urihttp://hdl.handle.net/11455/23826-
dc.description.abstractXanthomonas campestris pv. campestris (X. campestris)屬於葛蘭氏陰性菌,為兼具學術性及應用性之菌種。X. campestris為感染十字花科造成十字花科黑腐病之植物病原菌,屬世界性之病害。Xanthomonas campestris pv. campestris str. ATCC 33913之基因體定序已由巴西團隊完成且發表;本土菌株Xanthomonas campestris pv. campestris str. 17也由台灣團隊完成定序及基因註解。結構基因體學最終目標為解析整個基因體蛋白質之結構,同時希望以結構生物學的角度研究蛋白分子層次功能,與其他蛋白之間的交互作用。本研究挑選了X. campestris str. 17菌株16個蛋白質,其中XC1236、XC1258和XC1936為適合X-ray繞射法研究結構的蛋白質。XC1236為447個胺基酸組成,由生物資訊的方法得知為一乙醯穀胺酸(acetylglutamate kinase)激酶,其為磷酸化乙醯穀胺酸(N-acetyl-L-glutamate)。其重要性在於此為合成精胺酸(arginine) 不可或缺的反應。精胺酸為生物體內必需的化合物,除了係合成蛋白的原料外,更為生成尿素的中間物,參與如ornithine代謝反應的前驅物。XC1236目前致力於改善其晶體的大小及提高其繞射數據分辨率(resolution)。XC1936為240個胺基酸組成,由生物資訊的方法得知為一尿苷酸激酶。其機制為催化ATP的γ-磷酸轉移給UMP,形成UDP。由於XC1936與同功能的已知結構蛋白序列相似度相當高(55%),故我們嘗試以分子取代 (molecular replacement)法來解析其三級結構。XC1258由266個胺基酸組成, XC1258 在不同原核生物體皆有相似序列,由活性區域的保留序列推測為一具碳氮水解酶功能的蛋白。碳氮水解酶廣泛的參與非胜肽鏈的水解作用,生成一些重要的天然物,如biotin、auxin和一些抗生素的前驅物。但目前僅僅由比對序列得知其含一由Glu-Lys-Cys所組成的未知功能催化區,尚無足夠的結構證據來證明這個催化反應。於本研究我們得到XC1258有Selenomethionine標定的結晶(約1.75 Å),已進行多波長異常繞射數據收集,並得到初步的三級結構。接下來將繼續進行結構的建立與修正,以得到最完整的XC1258的三級結構。zh_TW
dc.description.abstractXanthomonas campestris pv. campestris is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris is a bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. The genome of Xanthomonas campestris pv. campestris str. ATCC 33913 was sequenced by ONSA/FAPBSP/Brazil group in 2002 and by an integrated structural and functional group in Taiwan in 2002. The goal of the structural genomics is to determine the three-dimensional structures of all proteins on a genome wide scale and to study the protein function from the perspective of structure biology at the same time. In this thesis we aim to identify and characterize the three-dimensional structures of several proteins in the X. campestris using X-ray crystallography. In the respect, three proteins, XC1236, XC1258, and XC1936, were found to be suitable for structural studies by X-ray crystallography from screening of 16 target proteins. XC1236 is a 447 amino acids protein that was annotated as an acetylglutamate kinase by a bioinformatics approach, which phosphorylates the N-acetyl-L-glutamate. This reaction is essential for the synthesis of arginine, which is an essential component of all living beings, a precursor for energy storage, and an intermediate in the production of urea and ornithine. We try to improve XC1236 crystal size and resolution. XC1936 is a 240 amino acids protein, which was annotated as a uridylate kinase by a bioinformatics approach. Its main function is to catalyze phosphate transfer from theγposition of ATP to UMP to form UDP. We try to use molecular replacement method to solve XC1936 structure. XC1258 is a 266 amino acids protein, and many sequences with high similarity with XC1258 were found in different prokaryotes. It was annotated as a CN-hydrolase from the conserved activity site sequence. The CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon-nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. We have obtained XC1258 Se-labeled crystals in a variety of forms suitable for X-ray diffraction studies (to at least 1.75 Å resolution). We have successfully applied the multiwavelength anomalous diffraction (MAD) method to determine its preliminary crystal structure. The structural refinement is currently udergoing.en_US
dc.description.tableofcontents致謝3 中文摘要4 Abstract 5 表目錄 10 圖目錄 10 縮寫檢索表 12 實驗儀器設備 13 第一章前言15 1-1、Xanthomonas campestris pv.campestris之結構基因體計畫簡介15 1-2、利用X-ray晶體繞射解析蛋白結構之特點17 1-3、研究動機及目的18 第二章 材料與方法19 2-1、挑選目標蛋白質19 2-2、構築蛋白質表現載體19 2-2-1、染色體DNA之抽取19 2-2-2、引子之設計與合成20 2-2-3、聚合酶連鎖反應(PCR, polymerase chain reaction)20 2-2-4、膠體電泳21 2-2-5、PCR product 之純化22 2-2-6、蛋白質表現系統22 2-2-7、質體DNA 之抽取23 2-2-8、PCR 產物與質體DNA 之限制酶作用23 2-2-9、DNA 之接合反應24 2-2-10、E.coli 勝任細胞之製備24 2-2-11、轉殖作用(Transformation)25 2-2-12、DNA 定序25 2-3、蛋白質之大量表現與純化 25 2-3-1、蛋白質大量表現之誘發條件25 2-3-2、SDS-PAGE25 2-3-3、蛋白質之大量表現27 2-3-4、蛋白質之純化28 2-3-5、蛋白質濃度測定30 2-3-6、蛋白質結晶實驗所需之蛋白質樣品製備31 2-4、利用X-ray 晶體繞射技術解析蛋白質之結構31 2-4-1、蛋白質結晶實驗31 2-4-2、結晶條件篩選32 2-4-3、大量結晶32 2-4-4、篩選合適之抗凍劑(Cryo-protectant)32 2-4-5、Native 蛋白晶體X-ray 晶體繞射數據收集及分析實驗33 2-4-6、相位(Phase)判定33 2-4-7、以X-ray 決定蛋白質之結構34 第三章 結果與討論35 3-1、目標蛋白質之選定35 3-2、XC123635 3-2-1、XC1236 之基因預測結果與功能註解分析35 3-2-2、XC1236 蛋白質表現載體之構築36 3-2-3、XC1236 蛋白質之大量表現與純化36 3-2-4、XC1236 之 X-ray 實驗結果討論37 3-3、XC125838 3-3-1、XC1258 之基因預測結果與功能註解分析38 3-3-2、XC1258 蛋白質表現載體之構築38 3-3-3、XC1258 蛋白質之大量表現與純化38 3-3-4、XC1258 之 X-ray 實驗結果討論39 3-4、XC193641 3-4-1、XC1936 之基因預測結果與功能註解分析41 3-4-2、XC1936 蛋白質表現載體之構築41 3-4-3、XC1936 蛋白質之大量表現與純化42 3-4-4、XC1936 之 X-ray 實驗結果討論42 第四章 參考文獻44 表目錄10 表3-1、挑選之目標蛋白質整理列表49 表3-2、引子設計50 表3-3、XC1236基本資料及蛋白家族51 表3-4、XC1258基本資料及蛋白家族52 表3-5、XC1936基本資料及蛋白家族53 表3-6、XC1258 Se-label晶體繞射數據54 表3-7、XC1936 Se-label晶體繞射數據55 圖目錄10 圖2-1、以X-ray晶體繞射決定蛋白質結構之實驗流程圖56 圖2-2、pET-32a之map57 圖2-3、蒸氣擴散法58 圖3-1、XC1236 –BLASTp之結果59 圖3-2、XC1236之定序結果60 圖3-3、XC1236之純化結果61 圖3-4、TEV切位及Selenomethionine 標定的XC1236 所得的Mass圖譜62 圖3-5、XC1236之結晶圖63 圖3-6、XC1236多聚體分析64 圖3-7、XC1236與其他乙醯穀胺酸激酶作序列比對65 圖3-8、XC1258 –BLASTp之結果66圖3-9、XC1258之定序結果67 圖3-10、XC1258之純化結果68 圖3-11、TEV切位及Selenomethionine 標定的XC1258 所得的Mass圖譜69 圖3-12、XC1258之結晶圖70 圖3-13、XC1258之Se-label X-ray晶體繞射圖譜71 圖3-14、XC1258與其他碳氮水解酶作序列比對72 圖3-15、XC1258與1EMS四聚體的比較73 圖3-16、XC1258單體結構73 圖3-17、XC1258、1F89與1EMS的superposition74 圖3-18、1F89、1EMZ和XC1258活性區域的比較74 圖3-19、XC1258多聚體分析75 圖3-20、XC1936 –BLASTp之結果76 圖3-21、XC1936(720bp)之定序結果77 圖3-22、XC1936之純化結果78 圖3-23、TEV切位及Selenomethionine 標定的XC1936 所得的Mass圖譜79 圖3-24、XC1936之結晶圖80 圖3-25、XC1936多聚體分析81 圖3-26、XC1936 與其它物種相同功能的蛋白做胺基酸序列比對82 圖3-27、XC1936之Se-label X-ray晶體繞射圖譜83zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.subjectmetabolicen_US
dc.subject代謝zh_TW
dc.titleX-ray繞射技術解析植物病原菌Xanthomonas campestris具重要代謝功能蛋白的結構研究zh_TW
dc.titleStructural studies of proteins with important metabolic functions from a plant pathogen Xanthomonas campestris using X-ray diffractionen_US
dc.typeThesis and Dissertationzh_TW
item.cerifentitytypePublications-
item.grantfulltextnone-
item.languageiso639-1en_US-
item.fulltextno fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
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