title: Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine.
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.
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Aubin-Tam
authors.forename: Adrian O
authors.initials: AO
authors.lastname: Olivares
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D021381:Protein Transport; D058767:Protein Unfolding
chemical_list: D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp