title: Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK.
abstract: Site-directed mutagenesis together with biochemical and biophysical techniques were used to probe effects of single-tryptophan-incorporated mutations on a bacterial molecular chaperone, Bacillus licheniformis DnaK (BlDnaK). Specifically, five phenylalanine residues (Phe(120), Phe(174), Phe(186), Phe(378) and Phe(396)) of BlDnaK were individually replaced by single tryptophans, thus creating site-specific probes for the fluorescence analysis of the protein. The steady-state ATPase activity for BlDnaK, F120W, F174W, F186W, F378W, and F396W was determined to be 76.01, 52.82, 25.32, 53.31, 58.84, and 47.53 nmol Pi/min/mg, respectively. Complementation test revealed that the single mutation at codons 120, 186, and 378 of the dnaK gene still allowed an Escherichia coli dnaK756-Ts strain to grow at a stringent temperature of 44°C. Simultaneous addition of co-chaperones and NR-peptide did not synergistically stimulate the ATPase activity of F174W and F396W, and these two proteins were unable to assist the refolding of GdnHCl-denatured luciferase. The heat-induced denaturation of all variants could be fitted adequately to a three-state model, in agreement with the observation for the wild-type protein. By CD spectral analysis, GdnHCl-induced unfolding transition for BlDnaK was 1.51 M corresponding to ΔG(N-U) of 1.69 kcal/mol; however, the transitions for mutant proteins were 1.07-1.55 M equivalent to ΔG(N-U) of 0.94-2.93 kcal/mol. The emission maximum of single-tryptophan-incorporated variants was in the range of 333.2-335.8 nm. Acrylamide quenching analysis showed that the mutant proteins had a dynamic quenching constant of 3.0-4.2 M(-1). Taken together, these results suggest that the molecular properties of BlDnaK have been significantly changed upon the individual replacement of selected phenylalanine residues by tryptophan.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City, Taiwan.
authors.forename: Bo-En
authors.initials: BE
authors.lastname: Chen
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D014364:Tryptophan; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis