title: A dual role of divalent metal ions in catalysis and folding of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1.
abstract: RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNH1) consists of an N-terminal domain with unknown function and a C-terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far- and near-UV CD spectra of Halo-RNH1 suggested that Halo-RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of Halo-RNH1 analyzed in the presence of salt and/or divalent metal ions by CD spectroscopy suggested that salt and divalent metal ions independently stabilize the protein and thereby facilitate folding. Divalent metal ions stabilize the protein probably by binding mainly to the active site and suppressing negative charge repulsions at this site. Salt stabilizes the protein probably by increasing hydrophobic interactions at the protein core and decreasing negative charge repulsions on the protein surface. Halo-RNH1 exhibited activity in the presence of divalent metal ions regardless of the presence or absence of 3 M NaCl. However, higher concentrations of divalent metal ions are required for activity in the absence of salt to facilitate folding. Thus, divalent metal ions play a dual role in catalysis and folding of Halo-RNH1. Construction of the Halo-RNH1 derivatives lacking an N- or C-terminal domain, followed by biochemical characterizations, indicated that an N-terminal domain is dispensable for stability, activity, folding, and substrate binding of Halo-RNH1.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Elias
authors.initials: E
authors.lastname: Tannous
authors.forename: Koji
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
keywords: Divalent metal ions; GdnHCl, guanidine hydrochloride; Halo-CTD, C-terminal domain (residues 69–199) of Halo-RNH1; Halo-NTD, N-terminal domain (residues 1–68) of Halo-RNH1; Halo-RNH1, RNase H1 from Halobacterium sp. NRC-1; Halobacterium sp. NRC-1; N-terminal domain; RNase H; RNase H, ribonuclease H; Salt-dependent folding