Journal
CELL
Volume 145, Issue 2, Pages 257-267Publisher
CELL PRESS
DOI: 10.1016/j.cell.2011.03.036
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Funding
- National Science Foundation [0643745]
- Howard Hughes Medical Institute
- National Institutes of Health [AI-15706, AI-82929]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0643745] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1118490] Funding Source: National Science Foundation
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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.
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