胃幽門螺旋桿菌染色體分離調控蛋白Spo0J晶體結構與功能之研究

Structural and Functional Investigation of Spo0J from Helicobacter pylori

許嘉恩(Chia-En Hsu)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2013年)

摘要

胃幽門螺旋桿菌是一種格蘭氏陰性微好氧的人類病原體，其感染胃黏膜層會導致胃炎及胃潰瘍等相關疾病。胃幽門螺旋桿菌26695編碼Hp1138基因，可轉譯出調控質體複製與分離的蛋白HpSpo0J，其為一種典型的螺旋轉螺旋(helix-turn-helix) DNA結合蛋白，具有專一性與parS DNA序列結合的能力。本論文計算出HpSpo0J與parS DNA結合之親和力並藉由X光繞射技術研究其晶體結構，有助於我們了解HpSpo0J在細菌質體分離過程中扮演的生物功能與分子機制。全長HpSpo0J與HpSpo0JN240（包含N端240個殘基）成功大量表現及純化，在水溶液中推測聚合型式分別為四聚體、二聚體。利用電泳遷移率（electrophoretic mobility shift）、螢光各向異性度（fluorescence anisotropy）進行DNA結合能力分析，並計算出HpSpo0JN240對parS DNA的解離常數為0.31±0.06 μM。此外，利用PEG3350為主沉澱劑成功得到HpSpo0JN240-parS complex晶體，並得到解析度為3.1Å繞射數據，其空間群 (space group) 為P21，晶格參數為a= 54.5 Å，b= 232.7 Å， c= 78.3 Å，β= 109.2°不對稱單元中含有四個分子，Vm值為2.5 Å3/Da，水溶液含量為51.5%。現階段，HpSpo0JN240-parS complex結構正在解析中。

關鍵字

胃幽門螺旋桿菌 ； 染色體分離調控蛋白

並列摘要

Helicobacter pylori is a spiral shape, microaerophilic Gram-negative human gastric pathogen. The colonization in stomach mucosa causes gastritis and peptic ulcer disease. The Hp1138 gene of Helicobacter pylori strain 26695 encodes a plasmid replication-partition related protein HpSpo0J, which is a classical helix-turn-helix DNA-binding protein that interacts with parS sequences (plasmid partition site of 16 bp). In order to understand the biological function and molecular mechanism of HpSpo0J involved in DNA segregation, we want to determine the DNA-binding affinity and the three-dimension structure of HpSpo0J-parS complex. In this study, full-length HpSpo0J and C-terminal truncated HpSpo0J (residues1-240, HpSpo0JN240) have been overexpressed and purified from E. coil, formed tetramer and dimer in solution. The DNA-binding activity of HpSpo0JN240 was performed by electrophoretic mobility shift assay and fluorescence anisotropy assay. The dissociation constant (Kd) of HpSpo0JN240-parS complex was calculated as 0.31±0.06 μM. HpSpo0JN240-parS complex was crystallized using PEG3350 as a precipitant and diffracted to 3.1Å resolution. HpSpo0JN240-parS complex crystal belongs to P21 space group with unit cell a= 54.5 Å, b= 232.7 Å, c= 78.3 Å, β= 109.2°. There are four molecules per asymmetric unit with Vm of 2.5 Å3/Da and solvent content of 51.5%. The structure determination of the HpSpo0JN240-parS complex is ongoing.

並列關鍵字

Helicobacter pylori ； Spo0J

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