title: Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases.
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.
authors: Department of Biology and.
authors: Vladimir
authors: V
authors: Baytshtok
authors: Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors: Tania A
authors: TA
authors: Baker
authors: Department of Biology and bobsauer@mit.edu.
authors: Robert T
authors: RT
authors: Sauer
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D059748:Proteolysis
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities
keywords: AAA+ protease; chaperone; molecular machine; protein unfolding