Structural and Functional Investigation of Spo0J from Helicobacter pylori

• Main Authors: Hsu, Chia-En, 許嘉恩
• Other Authors: Sun, Yuh-Ju
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/22684790739162918337

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 101 === 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.