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

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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/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. colidnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 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.

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Acknowledgments

We thank Prof. Hsien-Bin Huang of Institute of Molecular Biology, National Chung Cheng University for his thoughtful discussions to improve this manuscript. This work was supported by the research grants (NSC 95-2313-B-415-012-MY3 and NSC 97-2628-B-415-001-MY3) from National Science Council of Taiwan.

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Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, 600, Taiwan

    Wan-Chi Liang, Meng-Chun Chi, Hui-Ping Chang & Long-Liu Lin

  2. Department of Biochemical Science and Technology, National Chiayi University, 300 University Road, Chiayi, 600, Taiwan

    Min-Guan Lin

  3. Department of Food and Nutrition, Hungkuang University, Shalu, Taichung, Taiwan

    Hui-Yu Hu & Huei-Fen Lo

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  1. Wan-Chi Liang
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Correspondence to Huei-Fen Lo or Long-Liu Lin.

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Communicated by Wolfgang Buckel.

Wan-Chi Liang and Min-Guan Lin contributed equally to this work.

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Liang, WC., Lin, MG., Chi, MC. et al. Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis . Arch Microbiol 191, 583–593 (2009). https://doi.org/10.1007/s00203-009-0485-8

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  • DOI: https://doi.org/10.1007/s00203-009-0485-8

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