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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Chen, Bo-En Lin, Min-Guan Lo, Huei-Fen Wang, Tzu-Fan Chi, Meng-Chun Lin, Long-Liu |
| Description | Country affiliation: Taiwan Author Affiliation: Chen BE ( Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City, Taiwan.) |
| 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. |
| File Format | HTM / HTML |
| ISSN | 01418130 |
| Volume Number | 52 |
| e-ISSN | 18790003 |
| Journal | International Journal of Biological Macromolecules |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2013-01-01 |
| Publisher Place | Netherlands |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Biochemistry Adenosine Triphosphatases Chemistry Amino Acid Substitution Bacillus Enzymology Bacterial Proteins Models, Molecular Molecular Chaperones Tryptophan Genetics Metabolism Enzyme Stability Mutation, Missense Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Structural Biology Molecular Biology Biochemistry |
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