Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress‐induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat‐inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co‐operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.

Molecular chaperones of the Hsp7O class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the was initiated by addition of GrpE to 1.25 pM. Luciferase activity was measured using the Promega luciferase assay system and a Bio-Orbit luminometer (20). In Fig. 3, refolding was measured from a reisolated luciferase-DnaK-DnaJ complex formed in the presence of MgATP as described above, then separated from free nucleotide by a wash cycle consisting of three steps of a 1:20 dilution with buffer B, and concentration using a Centricon-30 filter device (Amicon). The original protein concentration was restored, and refolding was measured 1 hr after addition of nucleotides and GrpE, as indicated. To determine the percentage of luciferase aggregating, aliquots of each reaction mixture were taken 15 min after addition of nucleotides and GrpE and centrifuged (10 min at 30,000 x g), and the amount of luciferase in the pellet and supernatant fractions was quantified by SDS/PAGE and laser densitometry. Sedimentation of DnaJ and DnaK was negligible and was not affected by the addition of nucleotides or GrpE. In experiments containing competitive inhibitors for binding to DnaK or DnaJ, the luciferase-DnaK-DnaJ complex was initially formed as described above and separated from free nucleotide. After addition of the indicated concentrations of reduced carboxymethylated a-lactalbumin, a-casein, or DnaJ residues 1-127 to the reisolated complex, refolding was initiated by addition of GrpE and nucleotide. Refolding was measured after 1 hr.

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Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.