title: Mechanically Watching the ClpXP Proteolytic Machinery.
abstract: Energy-dependent protein degradation is studied through the dual bead ClpXP motility assay. Processing of folded proteins involves recognition, unfolding, translocation, and degradation stages. A dual optical trap, in a passive force-clamp geometry, exhibits bead-to-bead displacements that directly follow subprocesses underlying protein degradation. Discrete nanometer-scale displacements of the bead position reveal steps, dwells and pauses during the unfolding and translocation substeps. With a few structural modifications to the protease machinery and an engineered substrate, the assay represents a "chassis" for the measurement of a wide range of substrates and related machinery. The methods described faithfully record our assay as implemented, including substrate design, wet assay preparation, and the motility assay experiment protocol. The strategies herein permit adaptation of the ClpXP mechanical assay to a wide range of protein degradation systems.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Vanderbilt University, 308-A Olin Hall, VU Mailbox: PMB 351604, Nashville, TN, 37235, USA.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Cordova
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: Adrian O
authors.initials: AO
authors.lastname: Olivares
authors.affiliation: Department of Chemical and Biomolecular Engineering and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 308-A Olin Hall, VU Mailbox: PMB 351604, Nashville, TN, 37235, USA. matt.lang@vanderbilt.edu.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002850:Chromatography, Gel; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D052898:Optical Tweezers; D000072417:Protein Domains; D017510:Protein Folding; D055503:Protein Multimerization; D058767:Protein Unfolding; D059748:Proteolysis; D011994:Recombinant Proteins; D013194:Staining and Labeling; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp
keywords: AAA+ protease; ATPase; ClpXP; Dual trap; Optical tweezers; Passive force clamp; Proteasome; Single molecule degradation; Translocation; Unfolding