title: 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.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 600, Taiwan.
authors.forename: Wan-Chi
authors.initials: WC
authors.lastname: Liang
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Hu
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Hui-Ping
authors.initials: HP
authors.lastname: Chang
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases