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dc.contributor.authorMin-Shao Shihen_US
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dc.description.abstract過去的文獻指出FleQ 蛋白是控制細菌鞭毛基因表現的主要調控蛋白之一。它為一俱複雜功能的蛋白,可以做為一個 enhancer binding protein,也可以是一個 repressor protein。FleQ 蛋白的 N 端為 FleQ domain,中間為 AAA+/ATPase σ54 interaction domain,C 端為 helix-turn-helix DNA binding domain。另外,在文獻中也提到 FleQ 蛋白會受到 FleN 蛋白的調控而控制鞭毛的數量。FleN 蛋白為一個 ATPase,帶有一個特殊的 Walker A motif。在二級訊息傳遞分子 c-di-GMP 存在的情況下,FleN 蛋白上的 Walker A motif 會結合到 FleQ 蛋白的 N 端 domain (FleQ domain),進而調控 pel、psl 和 cdr 的基因表現,影響細菌合成鞭毛的蛋白表現,促使細菌產生生物膜 (biofilm)。 為了瞭解 FleQ 蛋白的作用方式,我們擬解析 FleQ 蛋白和與其功能相似的 FlrA 蛋白的結構。目前我們已經成功大量表現 S. maltophilia FleQ 全長蛋白、 N 端 FleQ domain 以及 Vibrio cholerae FlrA 全長和部分片段的蛋白。經由 X-光晶體繞射方法分析 SeMet-labeled SmFleQ domain 的蛋白晶體,解析度 (resolution) 可達 2.58 A,並且已成功解析出 SmFleQ domain 的結構。根據我們解析出的結構發現, SmFleQ domain 雖然被歸類為 REC domain,但是 SmFleQ domain 未帶有能接受磷酸根的 Asp,而且 SmFleQ domain 是以 α1-α5 相互作用形成二聚體,這些特點皆與典型的 REC domain 不同。本篇研究的結果對於非典型的 REC domain 有初步的了解。zh_TW
dc.description.abstractFleQ is a unique enhancer binding protein regulating many genes involved in the bacterial flagellar and exopolysaccharide biosynthesis. By responding to c-di-GMP, it helps modulating the transition between planktonic and biofilm lifestyles in some well studied bacteria. FleQ is a multi-domain activator belonging to the NifA/NtrC enhancer binding protein (EBP) family and comprises an N-terminal FleQ domain, an AAA+/ATPase α54 interaction domain, and a helix-turn-helix DNA binding domain. Unlike other bacterial two component signal transduction systems that transmit signals via the phosphorelay of the REC domain, FleQ is regulated through the binding of an anti-activator protein FleN to its N-terminal FleQ domain. In this manuscript, we reported the 2.58 A resolution crystal structure of the Stenotrophomonas maltophilia (S. maltophilia) FleQ domain. It is found to belong to the REC domain family, but adopts many atypical feature, including the absence of the crucial Asp residue in the acidic binding pocket to accept a phosphoryl group from a upstream cognate histidine kinase,?dislocated α2/α3/α4 helices, and a unique α1-α5 dimeric interface. These results form a basis to further understand how noncanonical REC domain transmit environmental signals in the bacterial kingdom.en_US
dc.description.tableofcontents致謝 I 中文摘要 II Abstract III 目錄 IV 表目錄 VII 圖目錄 VIII 縮寫檢索表 X 壹、 前言 1 一、 細菌的二級訊息傳遞子 cyclic-di-GMP 1 二、 Stenotrophomonas maltophilia 病原菌介紹 2 三、 Vibrio cholerae 介紹 3 四、 FleQ 蛋白介紹 3 貳、 材料與方法 5 一、 構築目標蛋白質及載體 5 (一)、 目標引子的設計 5 (二)、 萃取染色體DNA 5 (三)、 目標基因聚合酶連鎖反應 5 (四)、 以膠體電泳檢查目標蛋白片段 6 (五)、 純化基因片段 7 (六)、 Ligation-Independent cloning(LIC) 7 (七)、 勝任細胞之製備 9 (八)、 轉殖作用 (Transformation) 9 (九)、 目標基因定序 10 二、 點突變 (Site-Directed Mutagenesis) 11 三、 蛋白質之大量表現與純化 13 (一)、 目標蛋白質之大量表現 13 (二)、 純化目標蛋白 15 四、 利用 X-ray 晶體繞射技術解析蛋白質之三級結構 15 (一)、 蛋白質結晶條件篩選 15 (二)、 抗凍劑的選擇 16 (三)、 繞射數據收集 17 (四)、 決定相位角 17 (五)、 結構的建立與精算 19 五、 熱示差掃描實驗 (Differential Scanning Calorimeter, DSC) 19 參、 結果與討論 21 一、 標的蛋白之選擇 21 二、 標的蛋白之表現 21 (一)、 SmFleQ 21 1. 表現載體之構築、蛋白質表現與純化 21 2. 蛋白結晶與 X-ray 晶體繞射實驗 22 3. SmFleQFleQ 結構與分析 23 4. SmFleQFleQ 獨特的 REC domain interface 24 5. SmFleQ 之生物物理特性測量 24 (二)、 VcFlrA 25 1. 表現載體之構築、蛋白質表現與純化 25 2. 蛋白結晶條件篩選 25 3. VcFlrA 之生物物理特性測量 25 肆、 結論 27 伍、 參考文獻 28 表一、 SmFleQ 進行 PCR 所需的 Primer 序列 32 表二、 VcFleA 進行 PCR 所需的 Primer 序列 33 表三、 SmFleQ 進行點突變所需的 Oligonucleotides 序列 34 表四、 SeMet-SmFleQ1-143 之晶體繞射數據 35 圖一、 c-di-GMP生合成與訊息傳遞路徑 36 圖二、 c-di-GMP 調控 FleQ 蛋白結合 DNA 的能力 37 圖三、 c-di-GMP 與 FlrA 蛋白上的胺基酸 Arg 結合 38 圖四、 Ligation-Independent cloning (LIC) 的原理圖示 39 圖五、 蛋白質表現載體 pTBSG1 之序列圖 40 圖六、 蒸氣擴散法 41 圖七、 SmFleQ1-143 蛋白表現及純化結果 42 圖八、 SmFleQ1-493 蛋白表現及純化結果 43 圖九、 VcFlrA1-396 蛋白表現及純化結果 44 圖十、 SmFleQ 蛋白經過凝膠過濾法純化後的結果圖 45 圖十一、 SmFleQFleQ 蛋白晶體圖 46 圖十二、 VcFlrA1-396 蛋白晶體圖 47 圖十三、 SeMet-labeled SmFleQ1-143 晶體繞射圖譜 48 圖十四、 SmFleQ 測定 DSC 的結果 49 圖十五、 VcFlrA 測定 DSC 的結果以及 c-di-GMP 結合位之序列比對 50 圖十六、 SmFleQ 和 PaFleQ 之蛋白質序列比對 51 圖十七、 SmFleQFleQ 晶體結構 52 圖十八、 SmFleQ domain 和 PhoB、 DrrB、 Spo0F REC domains 的序列比對 53 圖十九、 SmFleQ domain 和 PhoB、 DrrB、 Spo0F REC domains 的結構比對 54 圖二十、 SmFleQ domain 二聚體的界面 55 圖二十一、 SmFleQF25A 和 SmFleQF25S 蛋白表現及純化結果 56 圖二十二、 SmFleQWT、 SmFleQF25A 和SmFleQF25S 經過凝膠過濾法後的結果 58zh_TW
dc.subjectcyclic di-GMPen_US
dc.titleStenotrophomonas maltophilia FleQ 及 Vibrio cholerae FlrA 蛋白的結構與功能分析zh_TW
dc.titleStructure and function studies of Stenotrophomonas maltophilia FleQ and Vibrio cholerae FlrAen_US
dc.typeThesis and Dissertationen_US
item.openairetypeThesis and Dissertation-
item.fulltextwith fulltext-
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