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研究生:胡惠喻
研究生(外文):Hui-Yu Hu
論文名稱:c-di-GMP如何增進LtmA與其同源DNA結合的結構研究
論文名稱(外文):How c-di-GMP enhances the binding of LtmA toward its cognate DNA sequences
指導教授:周三和
口試委員:陳金榜胡念仁
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:80
中文關鍵詞:二級訊息傳遞分子轉錄因子
外文關鍵詞:c-di-GMPtranscription factorsecondary messenger
相關次數:
  • 被引用被引用:0
  • 點閱點閱:60
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  • 下載下載:2
  • 收藏至我的研究室書目清單書目收藏:0
二級訊息傳遞分子cyclic-di-GMP (CDG)廣泛存在於細菌中,並參與調控許多重要的生物功能,包括致病性因子的產生、生物膜(biofilm)的合成以及細菌的移動。透過調控轉錄因子的活性,細菌能夠一次活化或抑制一群基因的表現。前人研究發現,許多調控細菌中重要生理活性的轉錄因子為CDG的受體蛋白,而CDG的結合會活化或抑制這些轉錄因子的活性。然而,目前對CDG是如何調控這些轉錄因子的詳細分子機制仍然不是很清楚。在2012年的文獻中指出恥垢分枝桿菌(Mycobacterium smegmatis)中的轉錄因子LtmA為CDG受體,此蛋白能辨識特定幾種脂質運輸和代謝基因的啟動子,當CDG結合到LtmA時,可增強此蛋白結合到其目標基因的能力。
為了瞭解CDG是如何影響LtmA的構形並增強其結合DNA的能力,本研究針對LtmA蛋白進行不同長度片段的構築及大量表現,並利用ITC及EMSA assay來研究,同時我們利用X-ray晶體繞射實驗來解析蛋白質結構,在養晶過程中加入CDG和/或DNA進行共結晶條件的篩選。最初有獲得LtmA9-184晶體且收到解析度為3.0 Å的X-ray繞射數據。目前已獲得native和SeMet-labeled LtmA9-184-CDG的晶體,且分別收到解析度為2.3和2.1 Å的X-ray繞射數據。接著利用異常散射(anomalous diffraction)解決相位角的問題,並成功解析出結構。從結構中得知,LtmA包含N端的DNA結合域(DNA-binding domain)和C端的配體結合域(ligand-binding domain),屬於類TetR家族的一員。然而,電子雲密度圖中卻無法偵測到CDG的存在。此外, 在LtmA與DNA的複合體結晶中,也收到解析度為2.6-Å的繞射數據,並順利解出結構,但也無法偵測到DNA的電子密度。未來將利用浸泡(soaking)或種晶(seeding)的方式嘗試解決配體問題,並且繼續篩選不同長度的DNA以期能獲得更高解析度的繞射數據。


Bis-(3''-5'')-cyclic dimeric guanosine monophosphate (c-di-GMP, CDG) is an important bacterial secondary messenger that is involved in the regulation of many critical processes including motility, biofilm formation and virulence. CDG exerts its regulatory function by directly binding to several types of receptor. A genetic screen in the non-pathogenic microorganism Mycobacterium smegmatis identified LtmA as a new CDG-responsive regulator, which is involved in lipid transport and metabolism. CDG could directly bind to LtmA and enhance its binding ability to its target gene. However, the reason for this enhancement is unclear. In addition, LtmA seems to lack the previously reported CDG binding motifs.
To elucidate how CDG binds to LtmA and enhance its DNA-binding ability, several structural and functional assays have been conducted. ITC and EMSA assay were carried out to detect binding between CDG-LtmA and DNA-LtmA. Initially we obtained the apo-form LtmA9-184 crystal and collected its X-ray diffraction data, which reached to a resolution of 3.0 Å. Currently, we have also obtained the native and SeMet-labeled co-crystals of LtmA with CDG. The X-ray diffraction data of native and SeMet-labeled co-crystals were collected to a resolution of 2.3 and 2.1 Å , respectively, and the initial structure of LtmA has been solved by SAD method (single wavelength anomalous dispersion). The results showed that LtmA contains a N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which are conserved in the TetR superfamily. However, we were unable to detect the presence of CDG in the electron density map. In addition, although we could obtain the crystals of LtmA in complex with DNA/CDG and collected their diffraction data to a 2.6 Å, we were unable to detect the presence of DNA/CDG in the electron density map. In the future, we will use soaking method at a higher ligand/protein ratio to obtain the LtmA-CDG co-crystal, and continue to screening DNA targets of varying-length for obtaining the LtmA-DNA-CDG ternary complex co-crystal of higher quality.


目次
中文摘要…………………………………………………………………………………I
Abstract………………………………………………………………………………….II
表目次………………………………………………………………………………....VII
圖目次………………………………………………………………………………..VIII
縮寫檢索表…………………………………………………………………………….IX
第一章、 前言 1
一、 細菌的二級訊息傳遞子c-di-GMP 1
二、Mycobacterium smegmatis LtmA 3
第二章、材料與方法 6
一、 構築蛋白質表現載體 6
(一) 染色體 DNA 的取得 6
(二) 引子設計與合成 6
(三) 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 7
(四) 膠體電泳 (agarose gel electrophoresis) 8
(五) PCR產物之純化 9
(六) 質體DNA之抽取 9
(七) Ligation-Independent cloning (LIC) 9
1. Insert的製備 10
2. Vector的製備 11
二、E.coli 勝任細胞 (competent cell) 之製備 12
三、轉殖作用 (Transformation) 13
四、PCR check 與 DNA 定序 13
五、蛋白質之大量表現與純化 15
(一) SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)的製備………….. 15
(二) 目標蛋白之大量表現 17
1. 蛋白質大量表現之誘發條件 17
2. 未標定之蛋白製備 18
3. Selenomethionine 標定蛋白之製備 18
(三) 目標蛋白質之初步純化 20
(四) 離子交換層析法 (ion exchange chromatography, IEC) 22
(五) 凝膠過濾法 (gel filtration) 22
(六) 蛋白濃度之測定 23
六、LtmA辨識之DNA設計 23
七、X-ray繞射晶體技術解析蛋白質之三級結構 24
(一) 蛋白質結晶實驗 24
(二) 結晶條件篩選 25
(三) 蛋白質與ligands/DNA的共結晶作用 (co-crystallization) 25
(四) 手動微調和大量結晶 26
(五) 篩選合適之抗凍劑(cryo-protectant) 26
(六) 收集繞射數據 (diffraction data collection) 27
(七) 相位角的決定 28
1. 同形取代法 (isomorphous replacement method) 28
2. 異常散射法 (anomalous dispersion method) 29
3. 分子置換法 (molecular replacement) 29
(八) 結構的建立 (model building) 與精算 (refinement) 30
八、恆溫滴定熱卡計(isothermal titration calorimetry, ITC) 30
九、電泳遷移漂移實驗(electrophoretic mobility shift assay, EMSA) 31
第三章、結果與討論 35
一、目標蛋白的選擇 35
二、標的蛋白之表現 35
(一) LtmA表現載體之構築、蛋白質表現與純化 35
(二) 蛋白結晶與 X-ray 晶體繞射實驗 36
1. LtmA 36
2. LtmA-CDG 37
3. LtmA-CDG-DNA 37
(三) LtmA晶體結構分析 38
1. LtmA9-184整體結構 38
2. CDG結合位置的預測 39
(四) 以ITC 分析LtmA蛋白質與CDG的結合強度 40
(五) 以EMSA分析LtmA與同源DNA的結合 41
第四章、結論 42
第五章、參考文獻 43

表目次
表 一、 LtmA辨識之DNA序列設計…………………………………………………50
表 二、自配Protein-DNA結晶用試劑……………………………………………….51
表 三、LtmA蛋白基本資料…………………………………………………………..54
表 四、MsLtmA 之晶體繞射數據……………………………………………………55
表 五、MsLtmA-CDG 之晶體繞射數據……………………………………………..56
表 六、LtmA-CDG-DNA之晶體繞射數據………………………………………….57

圖目次
圖 一、 c-di-GMP生合成與訊息傳遞路徑………………………………………….58
圖 二、 LtmA與其他TetR超家族的蛋白作序列比對…………………………….59
圖 三、與TFRs相互作用的小分子……………………………….…………………60
圖 四、 TetR家族的調控者們(regulators)展現不同ligand進入點………………….61
圖 五、 Ligation-Independent cloning (LIC) 的原理圖示…………………………...62
圖 六、 蛋白質表現載體 pTBSG1 之序列圖……………………………………….63
圖 七、 蒸氣擴散法…………………………………………………………………...64
圖 八、 恆溫滴定微卡計實驗………………………………………………………...65
圖 九、 Luminol 之化學發光機制…………………………………………………...66
圖 十、 其他蛋白質表現載體之序列圖……………………………………………...67
圖 十一、 LtmA9-184之大量純化PAGE圖……………………………………………68
圖 十二、 陰離子交換樹脂純化LtmA9-184的PAGE圖………………………………69
圖 十三、 以凝膠過濾法純化LtmA9-184 ……………………………………………..70
圖 十四、 LtmA晶體照片……………………………………………………….........71
圖 十五、 LtmA疊合圖.................................................................................................73
圖 十六、 LtmA9-184整體結構……………………………………………………….74
圖 十七、 LtmA9-184與FadR及Ms6564的結構比對……………………………….75
圖 十八、 Ms6564-DNA複合體結................................................................................76
圖 十九、 LtmA9-184-CDG位置預測圖...........................………………………………….77
圖 二十、 ITC測量LtmA與CDG結合情形的結果....................................................78
圖 二十一、 LtmA和DNA-29 nt 之EMSA實驗........................................................79




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