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研究生：	林奇歐 Lin, Chi-Ou
論文名稱：	大腸桿菌中鐵調控因子與其經亞硝基轉換後之生物無機化學研究 Identification and Characterization of Ferric Iron Uptake Regulatory Protein (Fur) and its Protein Bound Dinitrosyl Iron Complex (DNIC) in Escherichia coli
指導教授：	俞聖法 Yu, Sheng-Fa
共同指導教授:	黃守仁 Whang, Thou-Jen
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	98
中文關鍵詞：	雙亞硝基鐵化合物、鐵調控因子、轉錄因子、大腸桿菌、一氧化氮
外文關鍵詞：	Escherichia coli, ferric ion regulatory protein (Fur), nitrosylation, DiNitrosyl-Iron-Complexes (DNIC)
相關次數：	點閱：73 下載：1
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Fur(Ferric Iron Uptake Regulatory Protein)蛋白質被認為是在微生物系統中，其生理環境內，調控胞內鐵離子濃度的轉錄因子。
在本篇研究中，我們成功地利用重組基因，在大腸桿菌系統下過量表現Fur重組蛋白質，隨後使用鎳離子螯合管柱純化出Fur蛋白質，並藉由14% SDS-PAGE、Native-PAGE、LC-MS/MS、ICP-OES更進一步的確認Fur蛋白質特性。
Fur蛋白質加入二乙胺雙硝基化合物(diethylamine dinitric oxide, DEANO)，會使蛋白質中的鐵金屬生成雙亞硝基鐵化合物(Dinitrosyl Iron Complex, DNIC)。藉由電子順磁共振光譜儀偵測到雙亞硝基鐵化合物在gav=2.03的特殊訊號。
Fur蛋白質對基因序列“Fur-box”有極高的親合力(Kd=142.5 nM) ，亞硝基化的Fur蛋白質，則失去其對基因序列”Fur-box”的親和力，此外，這兩種狀態的蛋白質，在圓偏光二色性光譜偵測下，二級結構在組成比例上發生改變(亞硝基化後，Fur蛋白質的α-helix結構增加至3.3%；而有5.5%的β-sheet轉變成β-turn)。經由X光吸收近邊緣結構(X-ray Absorption Near-Edge Structures, XANES) 的數據顯示， Fur蛋白質活性中心呈現亞鐵離子二價組態，而亞硝基化Fur蛋白質活性中心，則是九個電子的雙亞硝基含鐵蛋白。重要的是，其配位對稱性在亞硝基化後產生明顯的改變。延伸X光吸收近邊緣細微結構 (Extended X-ray Absorption Fine Structure, EXAFS)顯示加入二乙胺雙硝基化合物後，Fur蛋白質中的鐵會從五配位的三角雙錐體或六配位的八面體結構轉變成四配位的四面體結構。藉由Spin label技術，在Wild Type-Fur上加入MTSL自由基標示於cysteine胺基酸上，以及利用點突變技術，設計突變Fur蛋白質以利MTSL自由基標示於目標位置上，以EPR觀測spin-spin interaction來推測活性中心鐵離子在Fur蛋白質的三度空間結構的位置。

A fur gene (447 bp) from the chromosomal DNA in Escherichia coli K12 were constructed into a pBAD--TOPOR vector (Invitrogen). After its transformation into Escherichia coli TOPO, we overexpressed and purified the recombinant Fur protein (MW=21.2kDa with His-tag) by nickel-charged histidine-binding resin column. Eluted fractions of Fur were further characterized by 14% SDS-PAGE, Native-page, LC-MS/MS, and elemental analysis via ICP-OES.

Active Fur performs as a homo-dimeric protein. Each of Fur monomer contains a ferrous iron, and a zinc ion. Its iron core could be further modulated to form a dinitrosyl iron complex (DNIC) after the treatment of gaseous NO or the NO donor of DEANO(diethylamine dinitric oxide). This protein bound DNIC is appeared with gav=2.03 observed in EPR spectroscopy.

Dimeric Fur can recognize a DNA element within Fur promoters with a 19-bp AT-rich palindromic sequence called a Fur-box. Here, we have determined that Fur protein can bind to the Fur-box with high affinity(Kd=142.5 nM) by EMSA(Electrophoretic Mobility Shift Assays) and fluorescence spectroscopy. After the treatment of NO, nitroylated Fur cannot bind with Fur-box.

Circular Dichroism spectra revealed that the secondary structure of nitrosylated Fur is different from that of Fur. More-turn structural component has been converted (5.5%) from part of -sheet structure. The iron core in Fur is presented as ferrous state whereas the nitrosylated Fur possess {Fe(NO)2}9. The symmetry of iron center in nitrosylated Fur protein is changed from trigonal bipyramidal or octahedral to tetrahedral geometry after treating with NO in X-ray Absorption Near-Edge Structures (XANES). Extended X-ray Absorption Fine Structure (EXAFS) results show that, the coordination numbers of iron center at the active site will be changed from five or six-coordinated environment with N/O ligands from His, Asp/Glu in distances of 2.16Å and 2.43Å to four-coordinated with N/O ligands from His, Asp/Glu upon the addition of DEANO. Finally, we attempt to incorporated MTSL(S-(2,2,5,5-tetramethyl-2,5-dihydro-1H- pyrrol-3-yl)methyl methanesulfonothioate) at appropriate site(s) with free cysteine or mutated by Cys in Fur, after nitrosylation, the spin-spin interaction detected by CW-EPR (Continuous Wave-Electron Paramagnetic Resonance) between the organic free radical and dinitrosyl iron complex within a distance in 5-15 Å should allow us to identify the location of the ferrous iron in the three dimensional structure of Fur.

中文摘要	I
ABSTRACT	III
誌謝	V
目錄	VIII
表目錄	XII
圖目錄	XIV
第一章	INTRODUCTION	1
1-1  序言	1
1-2  一氧化氮在生物系統中的來源及功能	2
1-3  一氧化氮	6
1-4  鐵調控因子氮白質(FERRIC UPTAKE REGULATOR, FUR)	9
1-5  FUR蛋白質的背景	11
1-6  表現載體	15
1-7  電子順磁共振	18
1-8  蛋白質自旋標記	19
1-9  X光吸收近邊緣結構(XANES)與延伸 X光吸收近邊緣細微結構(EXAFS)分析法	22
1-10  研究目的	25
第二章	實驗材料及方法	26
2.1  藥品與儀器	26
2.1.1  蛋白質來源	26
2.1.2  宿主細胞	26
2.1.3  藥品	26
2.1.4  儀器	27
2.2  實驗流程	35
2.3  蛋白質基因選殖	36
2.3.1  菌種培養	36
2.3.2  抽取染色體基因(Genomic DNA Extraction)	36
2.3.3  複製基因片段	37
2.3.4  純化聚合酶鏈式反應產物	38
2.3.5  轉型(Transformation)	39
2.4  蛋白質的大量表達(OVEREXPRSSION)	42
2.4.1  迴旋式恆溫震盪培養培養	42
2.4.2  發酵槽培養	42
2.5  蛋白質純化	43
2.5.1  破菌	44
2.5.2  蛋白質純化	44
2.5.3  過濾濃縮	45
2.6  表達蛋白質特性分析	46
2.6.1  SDS膠體電泳與NativePAGE電泳	46
2.6.2  膠體蛋白質消化與LC-MSMS分析	47
2.6.3  ICP-OES金屬含量分析	50
2.7  亞硝基化FUR蛋白質	50
2.8  圓偏光二色性光譜(CIRCULAR DICROISM, CD)	51
2.9  凝膠電泳測定FUR蛋白質與其作用DNA片段序列	51
2.10  螢光光譜儀測定FUR蛋白質及亞硝基化FUR蛋白質與其作用DNA片段序列	52
2.11  自由基標示(SPIN LABEL)	52
2.11.1  樣品製備	52
2.11.2  自由基標示	56
2.12  X-RAY吸收光譜	57
第三章	實驗結果與討論	58
3.1  表現系統PBAD-TOPO	58
3.2  表現蛋白質純化與特性分析	60
3.2.1  誘導試劑濃度測試及蛋白質純化	60
3.2.2  Native page	61
3.2.3  LC-MSMS	62
3.2.4  UV-vis光譜	63
3.2.5  ICP-OES	64
3.3  FUR蛋白質與亞硝基化FUR蛋白質之特性差異分析	65
3.3.1  EPR	66
3.3.2  圓偏光二色性光譜(Circular Dichroism, CD)	66
3.3.3  Fur蛋白質和亞硝基化Fur蛋白質對基因片段fur-box之作用	67
3.4  SPIN LABEL	70
3.5  XANES	72
3.6  EXAFS	72
3.7  實驗討論	75
第四章	結論	88
第五章	參考文獻	90
附錄一	93
附錄二	95
附錄三	97

                                    

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校外：2016-08-20公開

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