Involvement of Residues Asp8, Asn13, Glu145, Asp168, and Thr173 in the Chaperone Activity of a Recombinant DnaK from <i>Bacillus licheniformis</i>

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-Mg²⁺-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.

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

“…Mutations were created in the treA gene using QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the instructions of the supplier. After a thermocycling program described elsewhere (Lin et al 2011), the amplified products were treated with Dpn I at 37…”

Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK

“…To remove nucleotides bound onto wild-type and mutant BlDnaKs, the purified proteins were treated with alkaline phosphatase as described previously [18]. Nucleotide content of the sample supernatants was determined photometrically with the adsorption coefficient (ε 259 ) of 15.4 mM −1 cm −1 [19].…”