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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.

Author information

1
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
2
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
3
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; bobsauer@mit.edu tabaker@mit.edu.

Abstract

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.

KEYWORDS:

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

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