title: Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase.
abstract: We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre National de la Recherche Scientifique, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, 38044 Grenoble, France.|Institut Laue-Langevin, 38042 Grenoble, France.
authors.forename: Ziad
authors.initials: Z
authors.lastname: Ibrahim
authors.affiliation: Institut Laue-Langevin, 38042 Grenoble, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Martel
authors.affiliation: Institut Laue-Langevin, 38042 Grenoble, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Moulin
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre National de la Recherche Scientifique, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, 38044 Grenoble, France.
authors.forename: Henry S
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Institut Laue-Langevin, 38042 Grenoble, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Härtlein
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre National de la Recherche Scientifique, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, 38044 Grenoble, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Franzetti
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre National de la Recherche Scientifique, Institut de Biologie Structurale, 38044 Grenoble, France.|Centre à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, 38044 Grenoble, France.|Institut Laue-Langevin, 38042 Grenoble, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Gabel
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities / Q000737:chemistry* / Q000378:metabolism*; D001105:Archaea / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D053838:Scattering, Small Angle
chemical_list: D049452:Green Fluorescent Proteins; D000074183:ATPases Associated with Diverse Cellular Activities