Abstract
Bacterial DnaK belongs to the Hsp70 chaperone family, which plays a critical role in maintaining proteostasis by catalyzing protein folding, and is a proposed antibacterial target in the pathogen Mycobacterium tuberculosis. Here, we describe an experimental toolbox for evaluating inhibitors against the mycobacterial DnaK chaperone network: a coupled-enzymatic assay to monitor ATPase activity, a proteolytic cleavage assay to study DnaK conformational changes upon ligand addition, as well as a protein renaturation assay to assess chaperone function. For complete details on the use and execution of this protocol, please refer to Hosfelt et al. (2021).