title: Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein.
abstract: Molecular chaperones are an essential part of the machinery that avoids protein aggregation and misfolding in vivo. However, understanding the molecular basis of how chaperones prevent such undesirable interactions requires the conformational changes within substrate proteins to be probed during chaperone action. Here we use single-molecule fluorescence spectroscopy to investigate how the DnaJ-DnaK chaperone system alters the conformational distribution of the denatured substrate protein rhodanese. We find that in a first step the ATP-independent binding of DnaJ to denatured rhodanese results in a compact denatured ensemble of the substrate protein. The following ATP-dependent binding of multiple DnaK molecules, however, leads to a surprisingly large expansion of denatured rhodanese. Molecular simulations indicate that hard-core repulsion between the multiple DnaK molecules provides the underlying mechanism for disrupting even strong interactions within the substrate protein and preparing it for processing by downstream chaperone systems.
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; and.
authors.forename: Ruth
authors.initials: R
authors.lastname: Kellner
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; and.
authors.forename: Hagen
authors.initials: H
authors.lastname: Hofmann
authors.affiliation: Laboratoire de Biophysique Statistique, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Barducci
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; and.
authors.forename: Bengt
authors.initials: B
authors.lastname: Wunderlich
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; and schuler@bioc.uzh.ch.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D044085:Microfluidics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
chemical_list: C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D013884:Thiosulfate Sulfurtransferase; C063842:dnaK protein, E coli
keywords: FRET; Förster resonance energy transfer; Hsp40; Hsp70; protein folding