博碩士論文 107821018 詳細資訊




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姓名 黃正宏(Cheng-Hung Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 嗜酸熱硫化葉菌中DNA結合蛋白Sac10b之結構分析及其與DNA相互作用
(Crystal structure of DNA binding protein Sac10b from Sulfolobus acidocaldarius and its interaction with DNA)
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摘要(中) 中文摘要
在生物體中有一負責結合與調節遺傳物質的重要蛋白,稱之為DNA結合蛋白,顧名思義,它會透過不同方式與DNA結合,像是橋接、彎曲或纏繞。組蛋白是真核生物中常見的DNA結合蛋白,它以纏繞的方式包裹著DNA使DNA組織成緊密結實的染色質構造;與真核生物不同,古生菌沒有組蛋白而是利用各種小的類核蛋白,通過橋接或彎曲DNA分子的方式來組織基因組,Sac10b就是在古生菌中高度保守的核酸結合蛋白。本實驗中,使用各種方式更深入研究Sac10b蛋白質,從分析型超高速離心和圓二色譜分析結果顯示,兩種蛋白質Sac10b1、Sac10b2在溶液中均以熱穩定和耐酸的二聚體形式存在;從得到2.05-Å和1.70-Å解析度的Sac10b1、Sac10b2晶體結構中,分析出Sac10b1二聚體是以R58和F59為中心與另二聚體產生交互作用,再透過電子顯微鏡觀察,Sac10b1與DNA產生了協同結合形成一個完全橋接複合物,而Sac10b2卻無法和DNA形成完全橋接,可能就是缺少F59胺基酸。為了進一步證實R58和F59胺基酸的作用,從電子顯微鏡影像來看三個Sac10b1突變R58A、F59A和R58A/F59A與DNA作用,結果與Sac10b2相似,沒有觀察到與Sac10b1相同的協同結合完全橋接的行為。因此可以證實Sac10b透過橋接作用來組織DNA,R58和F59更是扮演關鍵角色的胺基酸。
摘要(英) Abstract
DNA-binding proteins that compact and regulate genetic materials are crucial in all organisms. Eukaryotic nuclear DNA is generally wrapped by histones into nucleosomes. Likewise, thermoacidophilic archaea utilize a variety of small nucleoid-associated proteins to organize the genome predominantly by bridging or bending the DNA molecule. Sac10b is a highly conserved nucleic acid-binding protein in archaea. Here we present two crystal structures of Sac10b1 and Sac10b2 from Sulfolobus acidocaldarius at 2.05-Å and 1.70-Å resolution, respectively. Each protein adopts a mixed α/β-fold with an extended β3-β4 hairpin. Analytical ultracentrifuge and circular dichroism results show that both proteins exist as thermal stable and acid tolerant dimers in solution. Electron microscopic images indicate that Sac10b1 and Sac10b2 can form condensed protein-DNA complexes by multiple bridged patches to different extents. Crystal packing analysis suggests that the dimer-dimer interactions, which involve the conservation of the interface residues, centered on R58 and F59 of Sac10b1, may account for the cooperative binding to DNA. The result is a completely bridged Sac10b1-DNA complex, which does not occur with the Sac10b2 homodimer due to the lack of an F59-equivalent residue. To further confirm the role of R58 and F59 residues, the EM images show that three Sac10b1 mutants R58A, F59A and R58A/F59A, similar to Sac10b2, result in the formation of intra-molecular bridging complexes but do not exhibit the cooperative binding behavior as Sac10b1, as no completely bridged DNA molecules were observed. A functional role for Sac10b in the organization and stabilization of chromosomal DNA through bridging interactions is suggested.
關鍵字(中) ★ 嗜酸熱硫化葉菌
★ DNA結合蛋白
★ X光繞射
★ 蛋白質結晶學
關鍵字(英) ★ Sulfolobus acidocaldarius
★ DNA-binding protein
★ X-ray crystallography
★ crystal structure
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菌種……………………………..………………………………………………………….1
1-1-1古菌域…………………..……………………………………………..……………..1
1-1-2古生菌硫化葉菌屬……..…………………………………………..………………..2
1-2 DNA結合蛋白……………………… ……………………………….………………...….3
1-2-1真核生物DNA結合蛋白……… …………………………….………………...….3
1-2-2古生菌DNA結合蛋白……...………………………….…….………………...….4
1-2-2-1 HMf蛋白質…………………………………….………………...……...….5
1-2-2-1 Alba蛋白質…………………………………….…………………...…...….5
1-3研究動機…………………………………………………………………………………...6
第二章 方法與材料…………………………………………………………………….……9
2-1實驗架構……………..………………………………….………………………………....9
2-2於載體(pET-21a)建構目標蛋白基因(Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A序列……...………………………..………10
2-2-1嗜酸熱硫化葉菌Sac10b1、Sac10b2基因體……………………….……….…….10
2-2-2聚合酶連鎖反應引子PCR Primer設計………………………………………..…11
2-2-3聚合酶連鎖反應Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A…...12
2-2-4膠體電泳確認目標基因片段………………………………………………..……..14
2-2-5建構表現質體………………………………………………..…………………….14
2-2-6轉型作用………………………………………………...……………………….…16
2-2-7菌落聚合酶鏈鎖反應………………………………………………………………16
2-2-8定序檢測 (Sequencing)……………………………………………………………18
2-3目大量蛋白質表現 (Protein Expression) ……...…………………….18
2-3-1測試最佳表現條件 (Time Course) …...…………………………………….18
2-3-2 SDS PAGE聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)………………………..18
2-4 Sac10b1、Sac10b2蛋白質大量表現…...….….…………………………………………19
2-5 Sac10b1、Sac10b2蛋白質純化……………………………………………………...…...19
2-5-1超音波破菌…………………………………………………………………………19
2-5-2加熱純化……………………………………………....……………………………20
2-5-3硫酸銨沉澱 (Ammonium Sulfate Precipitation)………………………..…..……..20
2-5-3-1鹽析………………………………...………………………………………...20
2-5-3-3硫酸銨 (Ammonia Sulfate) ……………………………………………….…20
2-5-4快速蛋白質液相層析儀 (Fast Protein Liquid Chromatography)純化………..…….21
2-5-4-1親和性純化法 (Immobilized Metal Affinity Chromatography) ……………21
2-5-4-2離子交換層析法 (Ion Exchange Chromatography) ………………….……..22
2-5-4-3膠體過濾法 (Size-exclusion Chromatography, Gel filtration) ……….……..22
2-6胺基酸序列點突變-Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A……....23
2-7蛋白質長晶………………………………………….………………………………....…23
2-7-1預長晶實驗 (Pre-crystallization Test) …………………….………………….…...23
2-7-2晶體條件篩選與晶體形成………………………….……………………………..24
2-8 X光繞射與結構解析…………………………………………………………………….25
2-8-1 X光繞射實驗……………….……………………….…………………………….25
2-8-2數據處理…………………………………………………………………………...26
2-8-3相位角……………………………………………………………….……………..26
2-8-4結構建立…………………………………………………………………………...26
2-9利用分析型超速離心機觀察蛋白質聚體情形……………………………...………….26
2-10 Sac10b1與Sac10b2耐熱耐酸測試……………………………………….….………….27
2-11利用電子顯微鏡觀察蛋白質與DNA結合……………………………………………27
第三章 實驗結果與討論…………………………………..……………………………….28
3-1建構目標基因Sac10b1與Sac10b2序列於pET-21a載體中………………………….28
3-2建構目標基因Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A序列於
pET-21a載體中. ……………...…………………...…………………...……………….28
3-3篩選適合蛋白質表現條件………………………………………...…………………….31
3-4 Sac10b蛋白質耐熱性…………………………………………………………….…….31
3-5利用陽離子交換樹脂管柱純化蛋白質…………………………………………………32
3-5-1 FPLC純化蛋白質Sac10b1、Sac10b2…………………………………………..32
3-5-2 FPLC純化蛋白質Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A..33
3-6硫酸銨沉降純化結果…………………...……………………….………………………34
3-7蛋白質長晶條件篩選……………………………………………………………………35
3-7-1 Sac10b1、Sac10b2長晶條件………………………………………………….…35
3-7-2 Sac10b1_F59A長晶條件…………………………………………...……………36
3-8蛋白質晶體上機與繞射數據……………………………………………………….……37
3-9蛋白質Sac10b1、Sac10b2結構……………………..…………………………………38
3-10蛋白質Sac10b1、Sac10b2與同源蛋白結構比較……….……………….....………..41
3-11蛋白質Sac10b二聚體-二聚體結構比較………...………………………………..…..42
3-12蛋白質Sac10b特性分析………………………………………………………..……..45
3-12-1分析型超速離心機……………….…………………………………..……….....45
3-12-2圓二色光譜Sac10b1與Sac10b2在溫度與pH值的耐受度…….….…46
3-13電子顯微鏡影像分析…………………………………………………………………..47
第四章 結論……………………….……………………………………………….….……49
參考文獻……………………………………………………………………………….……..50

圖目錄
圖一、親緣關係樹………………………………………………………………….…….……1
圖二、常見的組蛋白修飾………………………………………………….…………….……3
圖三、DNA結合蛋白特性示意圖……………………………………….…………….……..4
圖四、蛋白質Alba家族的胺基酸序列比對………………...………..…………………….…6
圖五、Sac10b實驗架構…………….……………………………………………….…..……..9
圖六、定序結果比對圖………………………………………………….………….…..……10
圖七、pET-21a全基因圖………………………..………………………………………..…..11
圖八、離子交換層析示意圖………………………….……………………………..……….22
圖九、座式晶盤養晶原理示意圖…………………………………..……………….……….25
圖十、Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59聚合酶鏈鎖反應產物……28
圖十一、Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A 菌落聚合酶鏈鎖反應產物…….………………………………………………………………….………….29
圖十二、a. Sac10b1 R58A序列比對………………………………………………….. …30
b. Sac10b1 F59A序列比對…………………………………………………..……30
c.Sac10b1 R58A/F59A序列比對………………………………………….……30
圖十三、Sac10b1蛋白質經過熱處理SDS-PAGE膠片圖……………..…..…31
圖十四、a.Sac10b1離子交換法層析圖………………………………………...……….….32
b. Sac10b2離子交換法層析圖………………………………………….…..….….32
c. Sac10b1、Sac10b2蛋白質純度的SDS-PAGE膠片圖…….……33
圖十五、a.Sac10b1_F59A離子交換法層析圖………………………………………….….33
b.Sac10b1_F59A SDS-PAGE膠片圖………….…………………….…..…34
圖十六、SDS-PAGE顯示硫酸銨沉降蛋白質Sac10b1_R58A主要析出濃度……………34
圖十七、β1-α1-β2-α2-β3-β4拓撲結構…................39
圖十八、Sac10b1和Sac10b2的整體結構和離子對相互作用………………….40
圖十九、Sac10b1、Sac10b2與同源蛋白結構比較疊圖…………………..42
圖二十、Sac10b蛋白二聚體-二聚體結構比較圖………………………………………….43
圖二十一、AUC沉降速度分布圖……………………………………………………….….45
圖二十二、Sac10b1與Sac10b2 CD光譜圖……………………………..…………....……46
圖二十三、Sac10b與PhiX174質體電子顯微鏡影像圖……………..….48

表目錄
表一、依DNA結合蛋白功能分類不同物種中的DNA結合蛋白…………....4
表二、已解出結構的Alba蛋白質……………………………………………………………5
表三、蛋白質Sac10b1基本性質…………………………………………………………...….7
表四、蛋白質Sac10b2基本性質…………………………………..………………….………8
表五、引子序列設計……………………………………………………………………...…12
表六、Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A聚合酶鏈鎖反應所需材料與反應條件參數…………………………………………...…13
a. 聚合酶鏈鎖反應所需材料………………………………….……………….……13
b. 聚合酶鏈鎖反應條件參數…………….………………………………………….13
表七、剪切反應所需材料……………………………………..……………………….……15
表八、黏合反應所需材料…………………………………..…………...............15
表九、Sac10b1_R58A、Sac10b1_F59A、Sac10b1_R58A_F59A菌落聚合酶鏈鎖反應所需材料與反應條件參數………………………………………………………..…….17
a. 菌落聚合酶鏈鎖反應所需材料………………………………….………….……17
b. 菌落聚合酶鏈鎖反應條件參數…………….…………………………………….17
表十、PCT試劑成分…………………………………………………………………….…..23
表十一、預長晶濃度測試表………………………………………………………………….24
表十二、a.成功解出結構之蛋白質Sac10b1長晶條件與晶體照片.…..35
b.成功解出結構之蛋白質Sac10b2長晶條件與晶體照片.……..35
表十三、成功解出結構之蛋白質Sac10b1_F59A長晶條件與晶體照片….36
表十四、Sac10b1、Sac10b2、Sac10b1_F59A的X-ray繞射數據…..……37
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指導教授 陳青諭(Chin-Yu Chen) 審核日期 2020-7-24
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