The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

In Escherichia coli, the DnaK/DnaJ/GrpE system plays a critical role in mediating protein refolding and buffering against protein aggregation due to environmental stress. The underlying mechanism remains unclear. In this work, we probe the activity of DnaK/DnaJ/GrpE system with single-molecule protein refolding assay using tandem repeats of titin immunoglobulin 27 (I27)8. We provide direct evidence that DnaK in apo- and ADP-bound state is predominantly a holdase, which kinetically stabilizes the polyprotein in its unfolded form. Binding of ATP relieves DnaK9s holding, allowing protein refolding. The presence of co-chaperone DnaJ and GrpE modulates this holding-release switching, possibly by altering DnaK9s nucleotide state. Our findings thus provide important insights to the molecular mechanism of DnaK/DnaJ/GrpE system.