Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis.
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.
Liang
Wan-Chi
WC
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 600, Taiwan.
Lin
Min-Guan
MG
Chi
Meng-Chun
MC
Hu
Hui-Yu
HY
Lo
Huei-Fen
HF
Chang
Hui-Ping
HP
Lin
Long-Liu
LL
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
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases