Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis

Abstract

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/DeltaC120, T86W/DeltaC249, and T86W/DeltaC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/DeltaC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44 degrees C. Except for T86W/DeltaC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/DeltaC120, and T86W/DeltaC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.

Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK

“…The overlapping complementary primers (Table 1) were designed to introduce the single point mutations at residues Phe 120 , Phe 174 , Phe 186 , Phe 378 and Phe 396 of BlDnaK in the plasmid pQE-BlDnaK [11]. Mutations were verified by DNA sequencing with dye terminator cycle sequencing kit and an automatic Perkin Elmer ABI Prism DNA sequencer.…”

“…Earlier, a recombinant Bacillus licheniformis DnaK (BlDnaK) was functionally expressed in E. coli M15 cells and allowed a heat sensitive strain (dnaK756-ts) to grow at the stringent temperature [11]. In the primary sequence of BlDnaK, the putative ATPase domain is located at the amino-terminal 364 residues and a putative SBD is spanned by residues 373-488, but the function of the carboxyl-terminal 134 residues remains to be elucidated.…”

Involvement of Residues Asp8, Asn13, Glu145, Asp168, and Thr173 in the Chaperone Activity of a Recombinant DnaK from <i>Bacillus licheniformis</i>

“…Mutations were introduced into pQE-Bl DnaK plasmid [Liang et al, 2009] using the QuikChange II site-directed mutagenesis kit according to the manufacturer's protocol. Two overlapping complementary primers containing the desired nucleotide changes were designed for each mutation ( table 3 ).…”

“…licheniformis DnaJ ( Bl DnaJ) and GrpE ( Bl grpE) were expressed and purified as described elsewhere [Liang et al, 2009].…”

“…Earlier, a recombinant Bacillus licheniformis DnaK ( Bl DnaK) was expressed in E. coli cells and allowed a dnakK756-ts mutant to grow at the stringent temperature [Liang et al, 2009]. Within the 622-residue sequence of Bl DnaK, the ATPase domain is located in the amino-terminal 364 residues, a SBD is spanned by residues 373-488, and the function of the carboxyl-terminal 134 residues remains unclear.…”