Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92200
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dc.contributor周三和zh_TW
dc.contributor.author楊詔貴zh_TW
dc.contributor.authorJauo-Guey Yangen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2014zh_TW
dc.date.accessioned2015-12-15T05:30:02Z-
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dc.identifier.urihttp://hdl.handle.net/11455/92200-
dc.description.abstract二級訊息傳導物質Cyclic guanosine 3',5'-monophosphate (cyclic GMP) 在細菌訊號傳遞中所扮演的功能在過去並非相當明確。然而在植物病原菌Xanthomonas campestris pv campestris (Xcc)的基因篩選實驗中發現,編碼XC_0250會轉譯出第三型nucleotidyl 環化酶,並合成cyclic GMP。在in vitro的實驗也證實純化出來的XC_0250具有合成cyclic GMP的能力。而其相連的基因XC_0249,被發現是一個帶有cyclic mononucleotide-binding (cNMP) domain以及 GGDEF diguanylate cyclase domain的蛋白。實驗發現,在cyclic GMP存在的條件下會提升XC_0249的cyclic di-GMP 合成能力。本篇研究我們解析了XC_0249 cNMP domain和cyclic GMP的複合體結構,並從結構的角度探討cyclic GMP和cyclic di-GMP是如何互相影響進而調控下游的生物生理機轉。而突變XC_0250或XC_0249的Xcc都將降低對植物的毒性以及降低生物膜的生合成。這樣的發現也因此連接了cGMP藉由影響cyclic di-GMP的合成調控整個訊息傳導路徑。除此之外,我們也嘗試著解析會和XC_0249的同源蛋白SM_0233交互作用的蛋白SM_RpfGD81E以及SM_RpfGD81E和SM_0233 GGDEF domain以及其cyclic di-GMP binding form的複合體結構,目前已初步得到SM_RpfGD81E-cyclic di-GMP的粗略結構。zh_TW
dc.description.abstractCyclic guanosine 3',5'-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signaling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified XC_0250 as a protein with a class III nucleotidyl cyclase domain, which is required for cyclic GMP synthesis. Purified XC_0250 is active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 contains a cyclic GMP binding site and a structure–function analysis, directed by determination of the crystal structure of the holo-complex and enzymatic assay confirm the cyclic GMP binding residues, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector XC_0249 that directly links cyclic GMP and cyclic di-GMP signaling. Besides, we also tried to determine the structure of SM_RpfGD81E which can interact with GGDEF domain of SM_0233, a XC_0249 homologous protein. We have obtained the crude full length SM_RpfGD81E crystal structure in complex with cyclic di-GMP. Further refinement is necessary to truly reveal its c-di-GMP binding mode.en_US
dc.description.tableofcontents目錄 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 第一章、 前言................................................................................................................1 第二章、 材料與方法....................................................................................................5 一、 轉位子突變與基因篩選........................................................................5 二、 辨識轉位子突變的插入點....................................................................5 三、 表現載體之構築....................................................................................5 (一)、本實驗室染色體 DNA 之取得...................................................5 (二)、目標引子的設計............................................................................6 (三)、聚合酶鏈鎖反應............................................................................7 (四)、膠體電泳........................................................................................8 (五)、DNA 之純化與萃取.....................................................................8 (六)、Vector及Insert的製備..................................................................8 (七)、Ligation-Independent cloning......................................................11 (八)、E. coli 勝任細胞之製備.............................................................12 (九)、轉殖作用......................................................................................12 (十)、質體DNA之抽取........................................................................13 (十一)、PCR check 與DNA 定序. .....................................................13 四、 環核苷酸環化酶實驗..........................................................................14 五、 RNA萃取與製備.................................................................................14 六、 蛋白質之大量表現與純化..................................................................14 (一)、蛋白質之大量表現......................................................................15 (二)、以親和性管柱進行目標蛋白的初步純化..................................15 (三)、TEV 蛋白水解酶作用................................................................16 (四)、使用凝膠過濾法純化目標蛋白..................................................16 七、利用X-ray 晶體繞射技術解析蛋白質之結構...................................17 (一)、蛋白質結晶條件篩選...............................................................17 (二)、protein 與ligand 的共結晶.......................................................18 (三)、protein 與protein 的共結晶.....................................................18 (四)、繞射數據的收集.........................................................................19 (五)、決定相位角.................................................................................19 (六)、結構的建立與精算.....................................................................19 八、點突變.................................................................................................20 九、利用區分熱穩定度來篩選適當的蛋白緩衝溶液.............................22 十、恆溫滴定微卡計實驗.........................................................................22 十一、熱示差掃描實驗.............................................................................23 十二、XC_0249酵素活性測試.................................................................23 第三章、 結果..............................................................................................................25 一、 基因篩檢證實在Xanthomonas中XC_0250是參與cyclic GMP生合成..........................................................................................................25 二、 XC_0250的cyclase domain具有合成cyclic GMP活性.....................25 三、 XC_0250對於最大致病性和生物膜形成是很重要的......................26 四、 XC_0249是一個會結合cyclic GMP的蛋白......................................27 五、 XC_0249的調節特性和XC_0250是有重疊的..................................28 六、 Cyclic GMP透過結合XC_0249的cyclic NMP-binding domain去調節cyclic di-GMP的訊息傳遞..............................................................28 七、 XC_0249所調控的生物膜合成與基因轉錄是仰賴它自身的DGC活性..........................................................................................................29 八、 SM_RpfGD81E具有能和cyclic di-GMP結合的能力...........................29 九、 SM_RpfGD81E結構探討.......................................................................30 十、 SM_0233的GGDEF domain與SM_RpfG之間的蛋白交互作用..31 十一、 根據熱穩定度篩選更適合的XC_0249 full-length以及SM_RpfGD81E緩衝液..................................................................................................32 第四章、 討論..............................................................................................................33 參考文獻.........................................................................................................................36 表目錄 表一、本研究各個蛋白結晶條件與晶體照片...............................................................42 表二、基因表現在XC_0249及XC_0250突變株中跟wild-type有差異的基因..........43 表三、利用ITC分析XC_0249WT以及其突變株和核苷酸的結合能力.......................51 表四、XC_0249 cNMP domain和cyclic GMP複合體的繞射數據與修正...................52 表五、SM_RpfGD81E 和 cyclic di-GMP 複合體繞射數據...........................................54 圖目錄 圖一、透過轉位子突變鑑定推測的鳥糞嘌呤環化酶...................................................55 圖二、XC_0250 cyclase domain和其他物種的胺基酸序列比對..................................56 圖三、單獨的XC_0250 CYC domain具有鳥糞嘌呤環化酶的活性.............................57 圖四、XC_0250酵素活性對於cyclic GMP生合成與生物膜形成的影響...................58 圖五、XC_0250突變株對於Xcc致病性和生物膜合成的影響....................................59 圖六、XC_0249蛋白的組成以及結合cyclic GMP能力的探討...................................60 圖七、ITC以及晶體結構分析顯示XC_0249是一個cyclic GMP結合蛋白................61 圖八、XC_0249 cNMP domain與cyclic GMP的結構圖...............................................62 圖九、XC_0249和E. coli CRP (EcCRP)的胺基酸序列比對.........................................63 圖十、利用ITC檢測XC_0249不同突變株和cyclic GMP的結合能力.......................64 圖十一、比較XC_0249以及XC_0250在轉錄組和表現型的影響..............................65 圖十二、Cyclic GMP濃度存在多寡與XC_0249酵素活性之關係..............................66 圖十三、Cyclic GMP的結合會影響XC_0249蛋白的雙鳥糞嘌呤環化酶活性,並且影響其他基因的表現.....................................................................................................67 圖十四、XC_0249的DGC活性會因為加入cyclic GMP而提升..................................68 圖十五、XC_0249突變株 (F73A, E90A)生物膜合成的能力比wild type來的差.......69 圖十六、SM_RpfGD81E和XC_RpfGD80E對於cyclic di-GMP的結合能力測試............70 圖十七、SM_RpfGD81E-cyclic di-GMP的初步結構.......................................................71 圖十八、SM_RpfGD81E和PmGH 的HD-GYP domain的疊合比較..............................72 圖十九、SM_RpfGD81E和PmGH HD-GYP domain binding site比較............................73 圖二十、SM_RpfGD81E HD-GYP domain binding site的疏水性鍵結............................74 圖二十一、SM_0233GGDEF+SM_RpfGWT 的膠體過濾法層析圖與SDS-PAGE圖......75 圖二十二、SM_RpfGD81E +SM_0233GGDEF 的膠體過濾法層析圖與SDS-PAGE圖.....................................................................................................................................76 圖二十三、SM_RpfGD81E +SM_0233GGDEF+cyclic di-GMP的晶體..............................77 圖二十四、SM_RpfGD81E 不同緩衝液的熱穩定度測試..............................................78 圖二十五、XC_0249 full length不同緩衝液的熱穩定度測試............................................79 圖二十六、Xcc中針對cyclic GMP和cyclic di-GMP所調控的生物膜合成以及致病性的模擬示意圖.............................................................................................................80 圖二十七、XC_0249 cNMP domain (紅色) 和 E. coli CRP (1G6N, 藍色) 的疊合比較.....................................................................................................................................81zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2015-01-22起公開。zh_TW
dc.subject環鳥糞嘌呤核苷酸zh_TW
dc.subject環雙鳥糞嘌呤核苷酸zh_TW
dc.subject二元傳遞訊息系統zh_TW
dc.subjectXC_0249zh_TW
dc.subjectXC_0250zh_TW
dc.subjectRpfGzh_TW
dc.subjectcyclic GMPen_US
dc.subjectcyclic di-GMPen_US
dc.subjecttwo component signal transductionen_US
dc.subjectXC_0249en_US
dc.subjectXC_0250en_US
dc.subjectRpfGen_US
dc.title從結構探討環鳥糞嘌呤核苷酸及環雙鳥糞嘌呤核苷酸控制的細菌訊息傳導機制zh_TW
dc.titleThe structral aspects of cyclic GMP and cyclic di-GMP-dependent bacterial signaling pathwaysen_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-01-22zh_TW
dc.date.openaccess2015-01-22-
item.languageiso639-1zh_TW-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.grantfulltextopen-
item.fulltextwith fulltext-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
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