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Cell. 2011 Apr 15;145(2):257-67. doi: 10.1016/j.cell.2011.03.036.

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine.

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  • 1Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Abstract

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:
21496645
[PubMed - indexed for MEDLINE]
PMCID:
PMC3108460
Free PMC Article
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