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Proc Natl Acad Sci U S A. 2017 Jul 19. pii: 201707794. doi: 10.1073/pnas.1707794114. [Epub ahead of print]

Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines.

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Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;


AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use single-molecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titinI27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titinI27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.


AAA+ motors; AAA+ proteases; directional unfolding; protein degradation

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