A gram-negative characteristic segment in Escherichia coli DnaK is essential for the ATP-dependent cooperative function with the co-chaperones DnaJ and GrpE |
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Authors: | Sugimoto Shinya Higashi Chihana Saruwatari Kozue Nakayama Jiro Sonomoto Kenji |
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Institution: | Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka, Japan. |
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Abstract: | We describe importance of the characteristic segment in ATPase domain of DnaK chaperone which is present in all gram-negative bacteria but is absent in all gram-positive bacteria. In vitro studies, ATPase activity, luciferase-refolding activity, and surface plasmon resonance analyses, demonstrated that a segment-deletion mutant DnaKDelta74-96 became defective in the cooperation with the co-chaperones DnaJ and GrpE. In addition, in vivo complementation assay showed that expression of DnaKDelta74-96 could not rescue the viability of Escherichia coli DeltadnaK mutant at 43 degrees C. Consequently, we suggest evolutionary significance for this DnaK ATPase domain segment in gram-negative bacteria towards the DnaK chaperone system. |
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Keywords: | BSA bovine serum albumin CD circular dichroism DnaKΔ74-96 segment (amino acids 74-96)-deletion mutant DnaK DnaKΔ75-97 segment (amino acids 75-97)-deletion mutant DnaK DTT dithiothreitol Gdn-HCl guanidine hydrochloride Hsp heat shock protein IPTG d-galactopyranoside" target="_blank">isopropyl-1-thio-β-d-galactopyranoside LB Luria-Bertani PCR polymerase chain reaction PMSF phenylmethylsulfonyl fluoride SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SPR surface plasmon resonance |
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