Optical Tweezers for Monitoring Unfolding by the Protease ClpXP

Abstract

The biological motor ClpXP pulls against a targeted protein until it mechanically unfolds and translocates the polypeptide into a chamber for degradation. We directly monitor degradation of model polyprotein substrates in dual-trap single molecule assays.

The Y9P Variant of the Titin I27 Module: Structural Determinants of Its Revisited Nanomechanics

“…Interestingly, when these variants were used as substrates for mitochondrial translocases, they showed a reduced rate of processing compared with I27wt when the unfolding process started at the N terminus (due to the stabilization in this region), but similar rates when the process started at the C terminus (due to a similar mechanical stability; Sato et al, 2005;Oguro et al, 2009;Yagawa et al, 2010). Therefore, our data indicate a close correlation between mechanical stability, as measured by SMFS, and the resistance to processing from these unfolding machineries (the AAA + -ATPase-based machineries are known to unfold their substrate proteins mechanically; Aubin-Tam et al, 2011;Maillard et al, 2011, Cordova et al, 2014, despite the differences in the pulling geometry between translocases and AFM (Wilcox et al, 2005). In any case, during its possible unfolding under physiological mechanical stress in the heart muscle, I27 would feel mechanical force applied to both of its termini, as occurs in SMFS, and not just at one end, as happens in the translocase assays.…”

“…This was surprising considering that this proline substitution was aimed at impeding the formation of one of the backbone H bonds in the module's mechanical clamp (Lu et al, 1998;Marszalek et al, 1999;Fowler et al, 2002), and therefore a reduced mechanical stability was expected. Interestingly, this variant, together with I27wt and other variants, has been used as a model substrate protein to examine the mechanism of protein unfolding by translocases and protein degradation machines (Kenniston et al, 2003(Kenniston et al, , 2004Sato et al, 2005;Feng and Lu, 2007;Oguro et al, 2009;Shin et al, 2009;Ruprecht et al, 2010;Yagawa et al, 2010;Cordova et al, 2014). The present study was initially aimed at understanding the structural basis of the surprising mechanical phenotype of I27Y9P reported previously (Li et al, 2000b).…”

Hinge–Linker Elements in the AAA+ Protein Unfoldase ClpX Mediate Intersubunit Communication, Assembly, and Mechanical Activity

“…Evidence of intersubunit communication is also observed in the enzyme's mechanical activity. Although the smallest translocation steps taken by ClpXP are ~1 nm in length, kinetic bursts of ATP hydrolysis appear to be responsible for steps of approximately 2, 3, and 4 nm [12][13] . Such communication could arise from contacts between neighboring large AAA+ domains and/or through the hinge-linkers that control the orientation of neighboring rigid bodies.…”

“…The ssrA tag initially binds to loops within the axial pore of the hexameric ClpX ring [8][9] . Conformational changes in the poredriven by ATP binding, hydrolysis, and product releasethen pull on the tag, eventually resulting in unfolding and stepwise translocation into ClpP for degradation [10][11][12][13] .…”

“…Mutant ClpX rings with only a single subunit capable of hydrolyzing ATP support low levels of ClpP degradation 17 , indicating that unfolding and translocation do not require sequential or concerted ATP hydrolysis. Although ClpX hydrolyzes many molecules of ATP while attempting to unfold a stable protein substrate, cooperative unfolding ultimately occurs as a consequence of a power stroke driven by a single hydrolysis event [10][11][12][13][18][19] . Robust unfolding requires coordination between the axial-pore loops of multiple ClpX subunits [20][21][22] .…”