E. coli ClpB is a Robust and Processive Protein Unfoldase

E. coli ClpB, and S. cerevisiae Hsp104, are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. Except for mitochondrial ClpB (Skd3), metazoans lack a ClpB/Hsp104 homologue. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through its axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate an unfolded loop at rates over 250 amino acids (aa) per second. Here we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ∼0.9 aa s-1 with a step-size of ∼60 amino acids. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered. ### Competing Interest Statement A.L.L. and J.B. are consultants for Nitrase Therapeutics.