博碩士論文 106821001 詳細資訊




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姓名 蔡孟儒(Meng-Ju Tsai)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 硫磺礦硫化葉菌程序性細胞死亡蛋白5晶體結構分析及其與DNA的相互作用
(Crystal Structure Analysis of Sulfolobus solfataricus Programmed Cell Death Protein 5 and Its Interaction with DNA)
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摘要(中) 中文摘要
程序性細胞死亡蛋白5 (PDCD5)在真核生物的細胞凋亡途徑中扮演著重要信號蛋白的角色,在現有的研究中,已知人類PDCD5蛋白中存在著三螺旋束和兩個游離的氮端區域,然而對於古生菌中PDCD5的研究仍尚不清楚。本研究中,我們鑑定了來自硫磺礦硫化葉菌的PDCD5同源蛋白(Sso-PDCD5),並且以1.55Å的高解析度呈現Sso-PDCD5的晶體結構,Sso-PDCD5為PDCD5同源蛋白中第一個解析出來的晶體結構,此蛋白質具有低柔韌性的緊密核心,氮端區域具有四個α-螺旋,以及具有彈性的碳端尾端。而位於碳端區域的色胺酸(W117)在激發螢光的實驗中發現色胺酸(W117)發散出的螢光可以被20bp的雙股DNA給遮蔽,螢光強度隨DNA濃度的增加而減少,這顯示Sso-PDCD5可以透過碳端尾端與DNA進行相互作用。在等溫滴定量熱的結果顯示碳端截斷蛋白(Sso-PDCD5_CTT,刪除K108-K118序列)顯著降低了與DNA結合的親和力,更進一步證明Sso-PDCD5利用動態的碳端尾端與雙股DNA進行結合。除此之外,電子顯微鏡(EM)的圖像顯示,Sso-PDCD5透過橋接的方式與雙股DNA相互作用結合。總而言之,經過結構以及生化方面的實驗數據表明Sso-PDCD5具有與雙股DNA結合的能力,是一種DNA結合蛋白。

關鍵字:硫磺礦硫化葉菌、程序性細胞死亡蛋白5、X光繞射、蛋白質結晶學、DNA結合蛋白
摘要(英) Abstract
The Programmed Cell Death protein 5 (PDCD5) is an important signal protein of apoptosis pathway in eukaryotes. In previous research, it is known there are triple-helix bundle and two dissociated N-terminal regions in human PDCD5 protein. However, the study of PDCD5 in hyperthermophile archaea remains unclear. Here, we identify a PDCD5 homolog from Sulfolobus solfataricus (Sso_PDCD5) and present the crystal structure of Sso_PDCD5 at a high resolution of 1.55 Å. This is the first crystal structure of a PDCD5 homolog to be solved, showing that the protein has a compact core of low flexibility with four alpha-helices at N-terminal region and a flexible unstructured C-terminal tail. The fluorescence of C-terminal tryptophan (W117) can be quenched by 20 bp double-strand DNA which indicates PDCD5 may interact with DNA by the C-terminal tail. The isothermal titration calorimety (ITC) results show C-terminal truncated protein (PDCD5_CTT, detection of K108-K118) significantly reduced the DNA-binding affinity, further demonstrated that the flexible C-terminus of Sso_PDCD5 involved in binding dsDNA. In addition, Sso_PDCD5 binds dsDNA through bridging interactions as shown in electron microscopy (EM) images. In conclusion, the structural and biochemical data suggest that Sso_PDCD5 may function as a DNA-binding protein.


Keywords: Sulfolobus solfataricus, the programmed cell death protein 5, crystal structure, X-ray crystallography, DNA-binding protein
關鍵字(中) ★ 硫磺礦硫化葉菌
★ 程序性細胞死亡蛋白5
★ X光繞射
★ 蛋白質結晶學
★ DNA結合蛋白
關鍵字(英) ★ Sulfolobus solfataricus
★ the programmed cell death protein 5
★ crystal structure
★ X-ray crystallography
★ DNA-binding protein
論文目次 目錄
中文摘要……………………………………………………………….……..………………..Ⅰ
英文摘要………………………………………………………………...…………….............Ⅱ
致謝………………………………………………………………………..………………….Ⅲ
目錄…………………………………………………………………………..…………...….Ⅳ
圖目錄….………………………………………………………………………...................Ⅷ
表目錄………………………………………………………………………………………....Ⅹ


第一章 緒論………………………………………………………………………...…1
1.1人類Programmed Cell Death protein 5(Human PDCD5)…………………………………1
1.2程序性細胞死亡(Programmed Cell Death)與細胞凋亡(Apoptosis)……………….……..3
1.3硫磺礦硫化葉菌…………………………………………………………………………...5
1.4古生菌 PDCD5 (Archaea PDCD5)………………………...……………………………...5
1.5蛋白質結晶學………………………………………..…………………………………….7
1.5.1結構生物學…………………………………………………………………………7
1.5.2 X光結晶學(X-ray crystallography)………………………………………………..7
1.5.3蛋白質晶體…………………………………………………………………………7
1.5.4布拉格定律(Bragg’s law)…………………………………………………………..9
1.5.5蛋白質晶體繞射原理………………………………………………………………9
1.5.6相位角決定方法…………………………………………………..……….……...10
1.5.6.1同型置換法(isomorphous replacement method)……………………..……10
1.5.6.2分子置換法(molecular replacement)………………………………..……..10
1.5.6.3非尋常散射法(anomalous dispersion method)…………….…………...….10
1.6研究動機與目的…………………………………………...……………………………..10
第二章 研究材料與方法……………………………………………………….……12
2.1 實驗架構設計…………………………………………………………………………....12
2.2 於載體(pET-21a)建構目標基因(Sso_PDCD5)序列……………………………………12
2.2.1 於硫磺礦硫化葉菌萃取目標基因(Sso_PDCD5)序列…………………………..12
2.3於載體( pET-21a )建構目標基因Sso-PDCD5_CTT基因序列…………………………13
2.3.1硫化葉菌Sso-PDCD5基因體…………………………………………………….13
2.3.2聚合酶連鎖反應引子PCR Primer設計………………………………………….13
2.3.3確認Sso-PDCD5基因序列於E.coli (DH5α)菌株中的正確性………………….15
2.3.4聚合酶鏈鎖反應(Polymerase Chain Reaction) Sso-PDCD5_CTT……………….15
2.3.5剪切反應實驗(Digestion)…………………………………………………………17
2.3.6目標基因(Sso-PDCD5_CTT)與載體(pET-21a)黏合反應(Ligation)…..…….….. 17
2.3.7製備勝任細胞 (Competent Cell)…………………………………………………18
2.3.8轉化作用(Transformation)…………..…………………………………….………18
2.3.9菌落聚合酶鏈鎖反應(Colony PCR)……………………………………….……..19
2.3.10瓊脂糖凝膠電泳反應( Agarose gel electrophoresis )…………………………...21
2.3.11定序檢測( Sequencing )………………………………………………………….21
2.4 Sso-PDCD5蛋白質表現( Protein Expression )………………………………………….22
2.4.1篩選最佳表現條件( Time Course )……………………………………………….22
2.4.2大量表現蛋白質…………………………………………………………………..23
2.5 Sso-PDCD5蛋白質純化(Protein Purification)…………………………………………23
2.5.1超音波震盪破菌後離心……………………………………………………...…...23
2.5.2目標蛋白(Sso-PDCD5)之耐熱性…………………………………………………24
2.5.3硫酸銨沉降 (Ammonium Sulfate Precipitation)…………………………………24
2.5.3.1鹽析及沉澱法……………………………………………………………...24
2.5.3.2鹽析(salting out)……………………………………………………………24
2.5.3.3硫酸銨(Ammonia Sulfate)…………………………………………………24
2.5.4管柱純化(Fast Protein Liquid Chromatography)………………………………….25
2.5.4.1親和性純化法(Immobilized Metal Affinity Chromatography)……………25
2.5.4.2離子交換層析法(Ion Exchange Chromatography)………………………..25
2.5.4.3膠體過濾法(Size-exclusion Chromatography, Gel filtration)……………..26
2.6胺基酸序列C端剃除(C-terminal truncation)…………………………………….……...27
2.7蛋白質晶體繞射……………………………………………………………………….…27
2.7.1預長晶實驗(Pre-crystallization Test)……………………………………….……..27
2.7.2機器手臂篩選長晶條件…………………………………………………………..29
2.7.3手動長晶…………………………………………………………………………..29
2.7.4 X光繞射實驗……………………………………………………………………..29
2.8電腦軟體數據分析……………………………………………………………………….30
2.8.1 HKL2000………………………………………………………………………….30
2.8.2 CCP4i……………………………………………………………………………...30
2.8.3 Coot………………………………………………………………………………..30
2.8.4 Pymol……………………………………………………………………………...30
2.9 Sso-PDCD5_WT與Sso-PDCD5_CTT耐熱耐酸測試………………………………….30
2.10 目標蛋白質對雙股DNA的結合程度…………………………………………………32
第三章 實驗結果與討論…………………………………………………………….33
3.1建構目標基因Sso-PDCD5與Sso-PDCD5_CTT序列在pET-21a載體中…………….33
3.2篩選適合表現目標蛋白的勝任細胞與生長條件……………………………………….35
3.3 Sso-PDCD5蛋白質耐熱性………………………………………...…………………….35
3.4硫酸銨沉降Ammonium sulfate precipitation……………………...…………………….36
3.5 FPLC ( Fast Protein Liquid Chromatography ) 膠體過濾法……………………………36
3.6長晶條件篩選與上機…………………………………………….………………………39
3.6.1 Sso-PDCD5_FL長晶條件……….………………………………………………..39
3.6.2 Sso-PDCD5_CTT長晶條件………………………………………………………40
3.7繞射數據……………………….…………………………………………………………41
3.8解開PDCD5蛋白質結構的方法比較(X-ray、NMR)…………………………….…….43
3.9 Sso-PDCD5晶體結構……………………………………………………………………43
3.10 Sso-PDCD5的電性分布…………………………………….………………...………..44
3.11 Sso-PDCD5結構細節………………………………………………….………………..45
3.12 Sso-PDCD5與其同源蛋白比較………………………………………………………..46
3.13 Sso-PDCD5與其同源蛋白結構間比較……………………………………….…..…...47
3.14 Sso-PDCD5功能……………………………………………………………….……….48
3.14.1 螢光光譜Fluorescence Quenching實驗………………………………………..48
3.14.2等溫低定熱分析儀(Isothermal titration calorimetry)……………………...……49
3.14.3圓二色光譜Sso-PDCD5在高溫與低pH值的耐受度…………………………51
3.15電子顯微鏡影像分析…………………………………………………………………...53
3.16 Programmed Cell Death相關蛋白研究………………………………….……………..54
3.17未來方向………………………………………………………………………………...56
3.17.1 Sso-PDCD5與人類PDCD5訊號途徑差異……………………………………56
3.17.2 Sso-PDCD5高溫測試與雙股DNA結合能力……….…………………………57
參考文獻……………………………………………………………………………….……..58
附錄…………………………………….……………………………………………………..60

圖目錄
圖一、人類PDCD5蛋白導致細胞凋亡路徑…………………………………………….……2
圖二、細胞受到刺激進行內在途徑細胞凋亡………………………..…………………….…4
圖三、內在途徑與外在途徑細胞凋亡的不同……………….………………………………..4
圖四、a.Human PDCD5與Sso- PDCD5 DNA序列相似度…………….……………..………6
b. Human PDCD5與Sso- PDCD5胺基酸序列相似度…………..…….………………6
圖五、坐式長晶過程(蒸氣擴散法)…………………………….……………………..………8
圖六、布拉格定律原理圖……………………………………………………………..……….9
圖七、Sso-PDCD5_FL與Sso-PDCD5_CTT實驗架構…………………………….……….12
圖八、根據pET-21a基因圖篩選適當限制酶切位…………….…………………….………13
圖九、根據pET-21a全基因圖中挑選出BamHI切位……….…………………….……….14
圖十、基因定序結果,確認完整的Sso-PDCD5基因序列保存於E.coli(DH5α)載體為pET-21a中…………………………………………………………………………………….15
圖十一、Sso-PDCD5_CTT基因建構轉化至E.coli,DH5α菌株的過程………………….…19
圖十二、定序結果之Sso-PDCD5_CTT目標基因……………………………………….….22
圖十三、生產大量表現目標蛋白質(Sso-PDCD5)流程圖…………………………….……23
圖十四、膠體過濾法(Gel filtration)純化原理…………………………………………….…27
圖十五、a.輕微沉澱……………………………………………………………………….….29
b.非典型重度晶體沉澱………………………………………………....................29
圖十六、主要二級結構於圓二色光譜測得之標準圖形…………………………………….31
圖十七、Isothermal titration calorimetry等溫滴定微量熱儀示意圖………………………..32
圖十八、Sso-PDCD5_CTT 聚合酶鏈鎖反應產物………………………………………….34
圖十九、Sso-PDCD5_CTT 菌落聚合酶鏈鎖反應產物…………………………………….34
圖二十、利用NCBI網站BLAST比對Sso-PDCD5_CTT基因序列結果…………………35
圖二十一、SDS-PAGE顯示不同濃度硫酸銨沉降蛋白質(a) Sso-PDCD5_FL與 (b) Sso-PDCD5_CTT之結果…………………………………………………………………….36
圖二十二、a. Sso-PDCD5_FL膠體過濾法層析圖………………….……………….......…..37
b. SDS-PAGE確認膠體過濾純化後,Sso-PDCD5_FL蛋白質的純度……......37
圖二十三、a. Sso-PDCD5_CTT膠體過濾法層析圖………………………......………...…..38
b. SDS-PAGE確認膠體過濾純化後,Sso-PDCD5_CTT蛋白質的純度……...37
圖二十四、Sso-PDCD5的整體結構…………………………………………….…………..44
圖二十五、 Sso-PDCD5靜電表面電位……………………………………………………..45
圖二十六、Sso-PDCD5結構細節………………………….……………………………..….45
圖二十七、參與Sso-PDCD5橫截面鹽橋的胺基酸……………………………………...…46
圖二十八、PDCD5同源蛋白多序列比對…………………………….……………………..47
圖二十九、PDCD5代表性同源蛋白的NMR結構………………….…………………...….47
圖三十、Sso-PDCD5與其他PDCD5同源蛋白結構疊加圖……………………………..….48
圖三十一、a. Sso-PDCD5色胺酸 (W117) 螢光強度受DNA作用而逐次減弱之光譜….49
b. 色胺酸 (W117) 相對螢光強度隨DNA濃度之變化關係圖………………..49
圖三十二、a. Sso-PDCD5_FL滴定雙股DNA的等溫滴定量熱法結果…….………...……50
b. Sso-PDCD5_CTT滴定滴定雙股DNA的等溫滴定量熱法結果……...……50
圖三十三、Sso-PDCD5_FL變溫 (a) 與變pH (b) 的圓二色光譜以及Sso-PDCD5_CTT變溫(c)的圓二色光譜…………………………………………………..…………………...….53
圖三十四、DNA結合蛋白與DNA作用時的構型示意圖………………………………....53
圖三十五、Sso-PDCD5_FL與DNA作用的電子顯微鏡影像圖…………….…..…...….….54

表目錄
表一、Sso-PDCD5_CTT聚合酶鏈鎖反應所需材料與反應條件參數……………………...16
a. 聚合酶鏈鎖反應所需材料……………………………………..…………………16
b. 聚合酶鏈鎖反應條件參數…………………………………....……….………….16
表二、剪切反應所需材料與比例…………………………………………………….………17
表三、黏合反應實驗步驟……………………………………………………………….……18
表四、Sso-PDCD5_CTT菌落聚合酶鏈鎖反應所需材料與反應條件參數...............………20
a. 菌落聚合酶鏈鎖反應所需材料………………………………….………….……20
b. 菌落聚合酶鏈鎖反應條件參數…………….…………………………………….20
表五、50X TAE Buffer配方…………………………………………………………….……21
表六、PCT試劑成分…………………………………………………………………….……28
表七、預長晶濃度測試表………………………………………………………….................28
表八、自動點晶儀篩選出Sso-PDCD5_FL長晶條件與晶體照片……………...…………..39
表九、手動優化Sso-PDCD5_FL蛋白質晶體的條件……………….……….…………..….40
表十、自動點晶儀篩選出Sso-PDCD5_CTT長晶條件與晶體照片…………………..……41
表十一、Sso-PDCD5_FL與Sso-PDCD5_CTT的X-ray繞射數據……………….……...….42
表十二、Programmed Cell Death相關蛋白及其別名與功能性質描述………….……..…..55
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指導教授 陳青諭(Chin-Yu Chen) 審核日期 2019-7-17
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