Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis.
The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions. The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains. The highly conserved amino-terminal portion is required for the ATPase activity. The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity. The mutant dnaK protein C[74], which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities. The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme. The autophosphorylating activity of the dnaK+, dnaK756, and C[74] polypeptides was activated at least 10-fold by the addition of CaCl2.
Cegielska
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A
Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
Georgopoulos
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C
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli