title: Involvement of residues Asp8, Asn13, Glu145, Asp168, and Thr173 in the chaperone activity of a recombinant DnaK from Bacillus licheniformis.
abstract: Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis DnaK (BlDnaK), a counterpart of Hsp70, and identified five different amino acids that might be responsible for maintaining ADP-Mg(2+)-Pi in the correct configuration at the ATP-binding cleft of the protein. As compared with wild-type BlDnaK, site-directed mutant proteins D8A, N13D, E145A, D168A, and T173A had a dramatic reduction in their chaperone activities. Complementation test revealed that the mutant proteins lost completely the ability to rescue the temperature-sensitive growth defect of Escherichia colidnaK756-ts. Wild-type BlDnak assisted the refolding of denatured firefly luciferase, whereas a significant decrease in this ability was observed for the mutant proteins. Simultaneous addition of B. licheniformis DnaJ, BlGrpE, and NR-peptide, did not synergistically stimulate the ATPase activity of D8A, E145A, D168A and T173A. Circular dichroism spectra were nearly identical for wild-type and mutant proteins, and they, except D8A, also exhibited a similar sensitivity towards temperature-induced denaturation. These results suggest that the selected residues are critical for the proper function of BlDnaK.
authors.affiliation: Departments of Biochemical Science and Biotechnology, National Chiayi University, Chiayi, Taiwan, ROC.
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.forename: Wan-Chi
authors.initials: WC
authors.lastname: Liang
authors.forename: Bo-En
authors.initials: BE
authors.lastname: Chen
authors.forename: Wei-Mou
authors.initials: WM
authors.lastname: Chou
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D017353:Gene Deletion; D005816:Genetic Complementation Test; D049409:Luciferases, Firefly / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis