Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori
| Autor: | Jian-Dong Huang, Rory M. Watt, Qing-Yu He, Ruiguang Ge, Julian A. Tanner, Hongzhe Sun, Xuesong Sun |
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| Rok vydání: | 2005 |
| Předmět: |
inorganic chemicals
Helicobacter pylori - metabolism Metal ions in aqueous solution Molecular Sequence Data Proteins - metabolism Mutant Storage medicine.disease_cause Biochemistry Serine Bacterial Proteins Nickel medicine Chelation Amino Acid Sequence Cloning Molecular Molecular Biology Escherichia coli Histidine chemistry.chemical_classification Helicobacter pylori Chemistry Nickel - metabolism Proteins Gene Expression Regulation Bacterial Cell Biology Amino acid Histidine-rich protein Dissociation constant Bacterial Proteins - metabolism Hpn Research Article Protein Binding |
| Zdroj: | Biochemical Journal. 393:285-293 |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | Hpn is a small cytoplasmic protein found in Helicobacter pylori, which binds Ni2+ ions with moderate affinity. Consisting of 60 amino acids, the protein is rich in histidine (28 residues, 46.7%), as well as glutamate, glycine and serine residues (in total 31.7%), and contains short repeating motifs. In the present study, we report the detailed biophysical characterization of the multimeric status and Ni2+-binding properties of purified recombinant Hpn under physiologically relevant conditions. The protein exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Using equilibrium dialysis, ICP-MS (inductively coupled plasma MS) and UV/visible spectroscopy, Hpn was found to bind five Ni2+ ions per monomer at pH 7.4, with a dissociation constant (Kd) of 7.1 μM. Importantly, Ni2+ binding to Hpn is reversible: metal is released either in the presence of a chelating ligand such as EDTA, or at a slightly acidic pH (pH for half dissociation, pH1/2 ∼6.3). Ni2+ binding induces conformational changes within the protein, increasing β-sheet and reducing α-helical content, from 22% to 37%, and 20% to 10% respectively. Growth curves of Escherichia coli BL21(DE3) both with and without the hpn gene performed under Ni2+ pressure clearly implied a role for Hpn to protect the cells from higher concentrations of external metal ions. Similarly, the accumulation of Ni2+ in these cells expressing Hpn from a plasmid was approx. 4-fold higher than in uninduced controls or control cultures that lacked the plasmid. Similarly, levels of Ni2+ in wild-type H. pylori 26695 cells were higher than those in H. pylori hpn-deletion mutant strains. Hpn may potentially serve multiple roles inside the bacterium: storage of Ni 2+ ions in a 'reservoir'; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+. © 2006 Biochemical Society. published_or_final_version |
| Databáze: | OpenAIRE |
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