Title

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

Translated Titles

Identification and Characterization of Ferric Iron Uptake Regulatory Protein (Fur) and its Protein Bound Dinitrosyl Iron Complex (DNIC) in Escherichia coli

DOI

10.6844/NCKU.2013.00255

Authors

林奇歐

Key Words

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 ; Escherichia coli ; ferric ion regulatory protein (Fur) ; nitrosylation ; DiNitrosyl-Iron-Complexes (DNIC)

PublicationName

成功大學化學系學位論文

Volume or Term/Year and Month of Publication

2013年

Academic Degree Category

碩士

Advisor

俞聖法

Content Language

繁體中文

Chinese Abstract

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。

English Abstract

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.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學系
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