ABSTRACT
ATPases associated with diverse cellular activities (AAA+) proteases power the maintenance of protein homeostasis by coupling ATP hydrolysis to mechanical protein unfolding, translocation, and ultimately degradation. Although ATPase activity drives a large portion of the mechanical work these molecular machines perform, how the peptidase contributes to the forceful denaturation and processive threading of substrates remains unknown. Here, using single-molecule optical trapping, we examine the mechanical activity of the caseinolytic peptidase P (ClpP) from Escherichia coli in the absence of a partner ATPase and in the presence of an activating small-molecule acyldepsipeptide. We demonstrate that ClpP grips protein substrate under mechanical loads exceeding 40 pN, which are greater than those observed for the AAA+ unfoldase ClpX and the AAA+ protease complexes ClpXP and ClpAP. We further characterize substrate-ClpP bond lifetimes and rupture forces under varying loads. We find that the resulting slip bond behavior does not depend on ClpP peptidase activity. In addition, we find that unloaded bond lifetimes between ClpP and protein substrate are on a timescale relevant to unfolding times (up to â¼160 s) for difficult to unfold model substrate proteins. These direct measurements of the substrate-peptidase bond under load define key properties required by AAA+ proteases to mechanically unfold and degrade protein substrates.
