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2007 | 54 | 3 | 509-522
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The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities

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Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaKMm) and the DnaK from the bacterium Escherichia coli (DnaKEc) were functionally similar when assayed in vitro but DnaKMm failed to substitute for DnaKEc in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaKMm and DnaKEc. DnaKMm showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaKEc. Furthermore, it was unable to negatively regulate the E. coli σ32 transcription factor level under heat shock conditions and poorly bound purified σ32, which is a native substrate of DnaKEc. These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaKMm showed some structural differences in the substrate-binding domains of DnaKMm and DnaKEc, which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaKMm was found preferably in high molecular mass oligomeric forms, contrary to DnaKEc. Oligomers of DnaKMm could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaKMm.
Physical description
  • Department of Biochemistry, University of Gdańsk, Gdańsk, Poland
  • Department of Biochemistry, University of Gdańsk, Gdańsk, Poland
  • Institute of Biochemistry, Faculty of Sciences, Universita Politecnica delle Marche, Ancona, Italy
  • Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdańsk, Gdańsk, Poland
  • Department of Biochemistry, University of Gdańsk, Gdańsk, Poland
  • Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland, USA
  • Department of Biochemistry, University of Gdańsk, Gdańsk, Poland
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