Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97750
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dc.contributor周三和zh_TW
dc.contributor.author陳采綸zh_TW
dc.contributor.authorTsai-Lun Chenen_US
dc.contributor.other生物化學研究所zh_TW
dc.date2019zh_TW
dc.date.accessioned2019-03-22T06:04:18Z-
dc.identifier.urihttp://hdl.handle.net/11455/97750-
dc.description.abstractC-di-GMP (cyclic dimeric guanosine 3',5'-monophosphate)是被發現廣泛存在於細菌中的二級訊號傳遞分子,並參與調控許多重要的生物功能,包括致病性因子的表現、生物膜(biofilm)的合成以及細菌的移動性等。2014年,Robert P. Ryan等人發現十字花科黑腐病菌(Xanthomonas campestris pv. Campestris, Xcc)中的YajQ為一種會和c-di-GMP結合的蛋白,並且發現YajQ可和LysR結合,進而調控細菌的致病性。本篇研究的菌種是產酶溶桿菌(Lysobacter enzymogenes, Le),其屬於十字花科黑腐病菌之亞種,並發現其合成的抗菌物質如heat-stable antifungal factor (HSAF),擁有高效的抗菌能力。2017年,南京農業大學錢教授團隊發現,LysR會與HSAF promoter (pHSAF)結合並調控HSAF的生合成。故本研究與錢教授團隊合作,為了瞭解產酶溶桿菌中,c-di-GMP如何調控LysR與YajQ之間的交互作用,並進一步影響LysR與pHSAF之間的結合。為了瞭解YajQ與LysR的構型,我們針對LysR進行多種不同長度的構築及大量表現,並在養晶時加入c-di-GMP及YajQ進行共結晶。目前已獲得native LysR86-286及Se-Met-LysR86-300的晶體,且分別收到解析度為2.6 Å 及2.7 Å的X-ray繞射數據。雖然目前尚未解出結構,但已利用small angle X-ray scattering (SAXS)的方式初步獲得YajQ、LysR86-300及YajQ-LysR86-300複合體的大略構型,發現LysR86-300在溶液中以二聚體形式存在,而YajQ是單體。然而LysR86-300與LysR1-388不一樣的是,在gel filtration中發現LysR1-388在溶液中是八聚體,故我們認為LysR1-388 N端的DNA binding domain對LysR的聚合態扮演重要的角色。為了探討c-di-GMP對於YajQ、LysR1-388與pHSAF的交互作用有何影響,我們利用surface plasmon resonance (SPR)的方式測得YajQ與LysR1-388之間的親和力為8.49 μM,而c-di-GMP會破壞YajQ與LysR1-388的結合。此外,也利用SPR測得LysR1-388與pHSAF的結合強度為44 nM,而YajQ-LysR1-388複合體與pHSAF的結合強度為29 nM,發現YajQ使LysR1-388與pHSAF之間的結合能力增強了50%。另外,加入100μM c-di-GMP會使LysR1-388與pHSAF之間的親和力下降,故我們認為c-di-GMP是破壞LysR1-388與YajQ之間的結合並使YajQ離開,而LysR1-388仍結合在DNA上,但在Xcc中c-di-GMP的存在會使LysR-YajQ-c-di-GMP複合體一起離開DNA,故我們發現Le與Xcc中LysR和YajQ之作用機制不一樣。zh_TW
dc.description.abstractC-di-GMP (cyclic dimeric guanosine 3',5'-monophosphate) is an important bacterial secondary messenger involved in the regulation of many critical processes including motility, biofilm formation and virulence. In 2014, Ryan's group found a novel c-di-GMP receptor protein YajQ in Xanthomonas campestris pv. Campestris (Xcc), and also discovered that some proteins such as LysR could interact with YajQ. In this study, we focused on the interaction between c-di-GMP, YajQ, LysR and pHSAF from Lysobacter enzymogenes (Le). Lysobacter can produce a new antibiotics such as heat-stable antifungal factor (HSAF), which has potential in controlling fungal diseases. In 2017, Qian's group found LysR could bind to HSAF promoter (pHSAF) and regulate the HSAF biosynthesis. For this reason, we cooperated with Qian's group to understand how c-di-GMP affects the interaction between LysR and YajQ, and further impact the binding of LysR with pHSAF. To elucidate how c-di-GMP affects the conformation of YajQ and YajQ-LysR complex, we constructed and overexpressed LysR of different lengths. In the meantime, we also crystallized YajQ with LysR and c-di-GMP, and got some crystals diffracted up to 2.7 Å. Although we are still unable to get the structure in detail yet, we have got the overall structure by using small angle X-ray scattering (SAXS) technique. Interestingly, while LysR86-300 forms a dimer in solution, we found that LysR1-388 adopts an octamer in solution by using gel filtration analysis. So, we assume that the N-terminal DNA binding domain of LysR1-388 may be important in determining the oligomerization of LysR protein. Furthermore, we used surface plasmon resonance (SPR) to measure the binding affinity of LysR with YajQ, and obtained a KD of 8.49 μM. In addition, c-di-GMP was also found to disrupt the interaction between LysR and YajQ. We also discovered that LysR had strong interaction with pHSAF, which revealed a KD of 44 nM, and the binding affinity between YajQ-LysR1-388 complex and pHSAF was about 29 nM. It appeared that YajQ increased the binding affinity of LysR1-388 with pHSAF by 50%. Our conclusion is different from Ryan's group's. We assume that c-di-GMP disrupts the interaction between YajQ and LysR1-388, but keeps LysR1-388 binding to pHSAF.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 表目次 v 圖目次 vi 縮寫檢索表 viii 第一章、前言 1 一、細菌的二級訊號傳遞分子c-di-GMP 1 二、Lysobacter enzymogenes產酶溶桿菌 2 三、新型c-di-GMP受體蛋白YajQ 3 四、YajQ的交互作用蛋白LysR 4 五、Heat-stable antifungal actor (HSAF) 5 第二章、材料與方法 7 一、構築目標蛋白質及表現載體 7 (一)、染色體DNA的取得 7 (二)、引子的設計 7 (三)、聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 8 (四)、膠體電泳 (Agarose gel electrophoresis) 8 (五)、目標基因片段之純化 9 (六)、質體DNA之萃取 9 (七)、Ligation-Independent cloning (LIC) 10 二、E.coli 勝任細胞(competent cell)之製備 12 三、轉殖作用(Transformation) 12 四、PCR check 與 DNA 定序 13 五、LeYajQ及LeLysR蛋白之大量表現及純化 13 (一)、目標蛋白之大量表現 13 (二)、目標蛋白之純化 15 (三)、凝膠過濾法(Gel filtration) 17 (四)、蛋白質濃度之測定 18 六、X-ray晶體繞射技術解析蛋白質之三級結構 18 (一)、蛋白質結晶實驗(Crystallization) 18 (二)、蛋白質與蛋白質/ligands之共結晶實驗(Co-crystallization) 19 (三)、蛋白質結晶條件之篩選 20 (四)、手動微調和大量結晶 20 (五)、繞射數據之收集(Diffraction data collection) 20 (六)、相位角的決定 21 (七)、分子模型的建立(Model Building)與精算(Refinement) 22 七、X光小角度散射(Small angle X-ray scattering, SAXS) 23 (一)、X光小角度散射(SAXS)23 (二)、SAXS實驗之原理 23 (三)、SAXS數據之收集與計算 23 八、YajQ及LysR蛋白之生物分子交互作用分析 24 (一)、恆溫滴定微卡計(Isothermal titration calorimetry, ITC) 24 (二)、表面電漿共振技術(Surface plasmon resonance, SPR) 25 第三章、結果與討論 28 一、LeYajQ及LeLysR基因片段長度之選擇 28 二、目標蛋白之大量表現 28 (一)、表現載體之構築、蛋白質表現及純化 28 (二)、蛋白質結晶實驗與繞射數據之收集 29 (三)、X光小角度散射實驗之結果 30 三、LeYajQ及LeLysR之生物分子交互作用分析結果 31 (一)、凝膠過濾法(Gel filtration) 31 (二)、恆溫滴定微卡計(Isothermal titration calorimetry, ITC) 32 (三)、表面電漿共振技術(Surface plasmon resonance, SPR) 33 第四章、結論 35 第五章、參考文獻 37zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2019-01-24起公開。zh_TW
dc.subject產酶溶桿菌zh_TW
dc.subjectLysobacter enzymogenesen_US
dc.titleLysobacter enzymogenes中的新型c-di-GMP受體蛋白YajQ與其交互作用蛋白LysR的功能與結構分析zh_TW
dc.titleStructural and functional studies of a novel c-di-GMP receptor protein YajQ and its binding protein LysR from Lysobacter enzymogenesen_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2019-01-24zh_TW
dc.date.openaccess2019-01-24-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypethesis and dissertation-
item.cerifentitytypePublications-
item.fulltextwith fulltext-
item.languageiso639-1zh_TW-
item.grantfulltextrestricted-
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