The genes coding for the Thermus thermophilus (Tth) homologues of the molecular chaperones DnaK and GrpE (DnaKTth and GrpETth) were cloned and expressed in Escherichia coli. The proteins were purified and their functional properties were assessed by equilibrium and transient kinetic methods. DnaKTth has an intrinsic ATPase activity of 3x10(-4) s-1 at 25 degreesC and 10x10(-4) s-1 at 75 degreesC under single turnover conditions. It binds the fluorescent nucleotide analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-diphosphate (MABA-ADP) with a dissociation constant (Kd) of 3 nM and ADP with a Kd of 47 nM at 25 degreesC. At 75 degreesC the affinities are decreased fivefold to 15 nM (MABA-ADP) and 280 nM (ADP). The kinetic constants for two-step binding of MABA-ADP and of ADP to DnaKTth were determined at 25 degreesC and 75 degreesC, respectively. GrpETth acts as a nucleotide-exchange factor on DnaKTth and accelerates the release of bound MABA-ADP significantly. This shows that the nucleotide-binding domain is functionally intact, and that the specific interaction of DnaKTth and GrpETth is mediating nucleotide exchange.A fluorescently labelled peptide that comprises a subsequence of the E. coli transcription factor sigma32 binds to nucleotide-free DnaKTth with a Kd of 4.9 microM. Displacement with unlabelled peptide yields a Kd of 5.0 microM for the unlabelled peptide. Thus the peptide-binding domain also appears to be functional.For the cellular chaperone function of DnaK, a coupling between nucleotide and peptide-binding domains is required. However, with DnaKTth in the ATP as well as in the ADP.Pi-state, peptide is bound and released within seconds. No correlation between ATP-binding or hydrolysis by DnaKTth and changes in the sigma32 peptide exchange rates could be detected. It thus appears that the DnaK system from Th. thermophilus has a different mechanism of coupling the nucleotide state to the fast and slow peptide exchange properties.
Copyright 1998 Academic Press Limited.