Xanthomonas campestris pv. campestris未知功能蛋白之大量表達與NMR結構分析

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胡玉真(Yu-Chen Hu) 查詢紙本館藏

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Xanthomonas campestris pv. campestris未知功能蛋白之大量表達與NMR結構分析
(Expression and structure determination of proteins of unknown function in Xanthomonas campestris pv. campestris)

★ Xanthomonas campestris pv. campestris未知功能蛋白XC847的晶體結構及功能分析

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摘要(中)

中文摘要
Xanthomonas campestris pv. campestris (Xcc)屬於格蘭氏陰性菌。為兼具學術性及應用性之菌種。XCC能分泌多種胞外蛋白，為研究格蘭氏陰性菌蛋白分泌之模式系統。其轉錄單位多以單基因方式為之，不具cAMP但以多元調控蛋白Clp (cAMP receptor protein-like protein)協助執行調控功能。XCC為一株感染十字花科造成十字花科黑腐病之植物病原菌，易生長於高溫多濕之環境，屬世界性之病害。且XCC所產生之多醣體Xanthan為重要之工業原料。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。
結構基因體學之目標期解出整個基因體之蛋白質結構。本研究希望以結構生物學的角度研究XCC蛋白之結構與功能。利用高磁場核磁共振儀(NMR)異核核磁共振實驗2D 15N-1H HSQC篩選Xcc str. 17菌株之18個目標蛋白質，並利用NMR方法決定兩蛋白質XC 975與XC 2382之初步蛋白質結構。XC 975由89個胺基酸組成，為未知功能蛋白，但推測其功能可能與逆境反應有關。因此其在COG蛋白質功能分類資料庫歸類為Stress-induced morphogen (COG0271)。利用收集的2D 15N-1H HSQC、3D HNCACB、3D CACB (CO)NH、HNCO、3D H(CC-CO)NH-TOCSY、3D (H)C(C-CO)NH-TOCSY等NMR光譜已完成大部分1H、15N以及13C之共振頻率判讀。利用TALOS軟體運算所得1HA, 13CA, 13CB, 13C’, 15N之化學位移與random coil的化學位移差並配合3D NOESY-15N-HSQC光譜分析得XC 975之二級結構的topology為αββααβ。XC 2382由127個胺基酸組成，此基因因常伴隨apaH表現，故基因命名為apaG，推測與apaH共同參與當細胞遇逆境時大量累積之Ap4N的代謝，但XC 2382實際的功能未知，為一未知功能蛋白。分析XC 2382與其同源蛋白所得的保守性序列中，含一與NAD及FAD結合之蛋白質特有的GXGVVGXXP保守性序列。而XC 2382在COG分類上為影響鎂離子與鈷離子運輸之蛋白質家族。XC 2382所得之2D 15N-1H HSQC光譜分散良好，由2D 15N-1H HSQC、3D HNCACB、3D CACB (CO)NH、HNCO、3D H(CC-CO)NH-TOCSY、3D (H)C(C-CO)NH-TOCSY等光譜已完成部分1H、15N以及13C之共振頻率判讀。

摘要(英)

Abstract
Xanthomonas campestris pv. campestris is a gram-negative bacterium that is phytophathogenic to crucifers such as Brassics and Arabidopsis and causes black rot. The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens. Structural genomics, which aims to determine the three-dimensional structures of all proteins on a genome wide scale. The genome of Xanthomonas campestris pv. campestris str. ATCC 33913 was sequenced by ONSA/FAPBSP/Brazil group in 2002 and that of Xanthomonas campestris pv. campestris str. 17 by an integrated structural and functional group in Taiwan in 2002 too. We try to understand, in structure terms, its pathogenicity to its host, its capability to produce xanthan gum, its protein secretion pathway, its different gene regulation behavior, and the proteins produced when it is under stress condition. Another important point about this structural genomics projects is to gain novel structural information. In this thesis we aim to identify and characterize the three-dimensional structures of several proteins in the XCC using high-resolution NMR techniques. In this respect, two XCC proteins, XC 975 and XC 2382, were selected for structural studies from screening 18 target proteins by 2D 15N-1HSQC NMR spectra. XC 975 codes for a hypothetical protein with a molecular mass of 9.7 kDa. A BLASTp search with this sequence revealed that most of the homologous sequences are annoted as hypothetical proteins. In COG database, XC 975 belongs to the family COG0271 named stress-induced morphogen. We have assigned nearly complete resonance of the 1H, 15N, 13C nuclei using 2D 15N -1H HSQC and triple resonance experiments including 3D HNCACB、3D CACB (CO)NH、HNCO、3D H(CC-CO)NH-TOCSY、3D(H)C(C-CO)NH-TOCSY etc. The XC 975 secondary structure topologyα β β α α β as calculated by the CSI program TALOS program using 1HA, 13CA, 13CB, 13C’ and 15N secondary chemical shifts and the complementary 3D NOESY-15N-HSQC spectrum data. XC 2382, with a molecular mass of 14.21 kDa, is named apaG because the gene is located in a multifunctional ksgA-apaG-apaH operon. The apaG expression is also tightly linked to apaH, suggesting that both gene products might be involved in the same biological function related to the metabolism of Ap4N. The unusual nucleotides (Ap4N) could be involved in the priming reaction of replication or synthesized as alarmones to signal the outset of cellular stress. XC 2382 has a highly conserved GXGXXG signature sequence, that is a pyrophosphate binding motif found in NAD- and FAD- binding proteins, suggesting that it may bind pyrophosphate or nucleotide phosphates. ApaG also shares sequence homology with CorD, a Salmonella typhimurium protein associated with Co2+ sensitivity and Mg2+ homeostasis. The high degree of sequence conservation among ApaG homologs in bacteria indicated that it carries out some important biological functions. We have also assigned most of the resonances of the 1H, 15N, 13C nuclei using similar NMR experiments as described above.

關鍵字(中)

★ 蛋白質結構
★ 結構基因體
★ 未知功能蛋白
★ 核磁共振
★ Xanthomonas campestris pv. campestris

關鍵字(英)

★ structural genomics
★ Xanthomonas campestris pv. campestris
★ NMR
★ protein structure
★ unknown function protein

論文目次

目錄
第一章前言
1-1、Xanthomonas campestris pv.campestris 之結構基因體計畫簡介 1
1-2、利用高磁場核磁共振儀研究蛋白質結構之簡介 3
1-3、研究目的 3
第二章材料與方法
2-1、目標蛋白質之選定 5
2-2、蛋白質表現載體之構築
2-2-1、染色體DNA 之抽取 5
2-2-2、引子之設計與合成 6
2-2-3、聚合酶連鎖反應 7
2-2-4、膠體電泳 8
2-2-5、DNA 之純化8
2-2-6、融合蛋白表現系統 9
2-2-7、質體DNA 之抽取 9
2-2-8、PCR 產物與質體DNA 之限制酶作用 9
2-2-9、DNA 之接和反應 10
2-2-10、E.col 勝任細胞之製備 10
2-2-11、轉形作用 11
2-2-12、DNA 之定序 11
2-3、蛋白質之大量表現與純化
2-3-1、蛋白質大量表現之誘發條件 11
2-3-2、SDS-PAGE 11
2-3-3、蛋白質之大量表現
2-3-3-1、15N 標定之蛋白質製備 13
2-3-3-2、15N, 13C 標定之蛋白質製備 13
2-3-3-3、15N, partial 13C 標定之蛋白質製備 13
2-3-4、蛋白質濃度測定 14
2-3-5、結合蛋白與蛋白脢之反應條件試驗 14
2-3-6、蛋白質之純化
2-3-6-1、MBP 融合蛋白之純化 16
2-3-6-2、His-tag 融合蛋白之純化 16
2-3-6-3、GST 融合蛋白之純化 17
2-3-7、分子量之測定 18
2-3-8、NMR 實驗所需之蛋白質樣品製備 18
2-4、利用核磁共振方法研究蛋白質之結構
2-4-1、核磁共振實驗 18
2-4-2、決定蛋白質之結構 19
2-4-3、NMR 圖譜判讀 19
2-4-4、雙面角限制條件 21
2-4-5、距離限制條件 21
2-4-6、氫鍵限制條件 22
第三章結果與討論
3-1、目標蛋白質之選定 23
3-2、XC 975
3-2-1、XC975 之基因預測結果與功能註解分析 23
3-2-2、XC 975 蛋白質表現載體之構築 25
3-2-3、XC 975 蛋白質之大量表現與純化 25
3-2-4、XC 975 蛋白質之NMR 圖譜分析 26
3-2-5、XC 975 之實驗結果討論 27
3-3、XC 2382
3-3-1、XC 2382 之基因預測結果與功能註解分析 29
3-3-2、XC 2382 蛋白質表現載體之構築 30
3-3-3、XC 2382 蛋白質之大量表現與純化 31
3-3-4、XC 2382 質蛋白之NMR 圖譜分析 31
3-3-5、XC 2382 之實驗結果討論 32
第四章參考文獻 34

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指導教授

周三和(Shan-Ho Chou)

審核日期

2004-7-24

推文