title: Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori.
abstract: 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 (K(d)) of 7.1 microM. 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 approximately 6.3). Ni2+ binding induces conformational changes within the protein, increasing beta-sheet and reducing alpha-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 Ni2+ ions in a 'reservoir'; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+.
authors.affiliation: Department of Chemistry and Open Laboratory of Chemical Biology, The University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China.
authors.forename: Ruiguang
authors.initials: R
authors.lastname: Ge
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Xuesong
authors.initials: X
authors.lastname: Sun
authors.forename: Julian A
authors.initials: JA
authors.lastname: Tanner
authors.forename: Qing-Yu
authors.initials: QY
authors.lastname: He
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Huang
authors.forename: Hongzhe
authors.initials: H
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000378:metabolism*; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D000099053:Histidine-Rich Glycoprotein
chemical_list: D001426:Bacterial Proteins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D009532:Nickel