title: Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions.
abstract: Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity ( 1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.
authors.affiliation: Department of Chemistry, Faculty of Science and Technology, Meijo University, Nagoya, Aichi, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Hibino
authors.forename: N
authors.initials: N
authors.lastname: Kaku
authors.forename: H
authors.initials: H
authors.lastname: Yoshikawa
authors.forename: T
authors.initials: T
authors.lastname: Takabe
authors.forename: T
authors.initials: T
authors.lastname: Takabe
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003300:Copper / Q000378:metabolism; D000458:Cyanobacteria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D012492:Salts / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D012492:Salts; D003300:Copper; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli