Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases
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Baytshtok-2015-Assaying the kinetic.pdf
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Author(s)
Baytshtok, Vladimir
Baker, Tania
Sauer, Robert T
Date Issued
April 2015
Journal
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences (U.S.)
Citation
Baytshtok, Vladimir, Tania A. Baker, and Robert T. Sauer. “Assaying the Kinetics of Protein Denaturation Catalyzed by AAA+ Unfolding Machines and Proteases.” Proc Natl Acad Sci USA 112, no. 17 (April 13, 2015): 5377–5382.
Version
Final published version
Abstract
ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1073/pnas.1505881112
