title: Hsp70 biases the folding pathways of client proteins.
abstract: The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8;
authors.forename: Ashok
authors.initials: A
authors.lastname: Sekhar
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel;
authors.forename: Rina
authors.initials: R
authors.lastname: Rosenzweig
authors.affiliation: Département de Chimie, École Normale Supérieure (ENS)-Paris Sciences et Lettres (PSL) Research University, 75005 Paris, France; Laboratoire des Biomolécules (LBM), Sorbonnes Universités, Université Pierre et Marie Curie (UPMC), Université Paris 06, 75005 Paris, France; UMR 7203 LBM, CNRS, 75005 Paris, France;
authors.forename: Guillaume
authors.identifier: 0000-0003-4398-0320
authors.initials: G
authors.lastname: Bouvignies
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Biochemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Department of Chemistry, The University of Toronto, Toronto, ON, Canada M5S 1A8; Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 kay@pound.med.utoronto.ca.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D017510:Protein Folding; D035321:Telomeric Repeat Binding Protein 1 / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D018840:HSP70 Heat-Shock Proteins; D035321:Telomeric Repeat Binding Protein 1
keywords: Hsp70; PRE; excited states; molecular chaperones; protein folding