Abstract
AimsThe aim of this study was to characterize the recombinant Bacillus licheniformis DnaK (BlDnaK) and to enhance the understandings of allosteric regulation in this protein by deletion mutations.Methods and ResultsThe heat shock protein 70, heat shock protein 40 (DnaJ), and nucleotide exchange factor (GrpE) genes of B. licheniformis were overexpressed in Escherichia coli. The purified BlDnaK had a Vmax of 32.5 nmol Pi/min and a KM of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756‐ts mutant to grow at 44oC. Simultaneous addition of DnaJ and GrpE synergistically stimulates the ATPase activity of BlDnaK by 11.7‐fold. A T76W mutant and three C‐terminally truncated BlDnaKs were also constructed to evaluate the role of up to C‐terminal 255 amino acids in allosteric regulation. The specific ATPase activity for BlDnaK, T76W, T76W/δC120, T76W/δC249, and T76W/δC255 was 1.45, 1.06, 2.32, 6.43, and 1.81 nmol/Pi/min/mg, respectively. Measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acids in the C‐terminally truncated BlDnaKs. The temperature‐dependent signal in the far‐UV region for T76W was consistent with that of BlDnaK, but the C‐terminally truncated mutants showed a higher sensitivity towards temperature‐induced denaturation.ConclusionsThis study provides evidence that BlDnaK has biochemical and molecular chaperone properties of the Hsp70/DnaK family. Our preliminary results also showed that the ATPase activity of the variants was stimulated differently from wild‐type BlDnaK in presence of BlGrpE.
Published Version
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