title: Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy.
abstract: DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase.13C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wiegand
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cadalbert
authors.affiliation: Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France.|CRM2, UMR 7036 CNRS/, Université de Lorraine, Faculté des Sciences et Technologies, BP 70239, Boulevard des Aiguillettes, 54506, Vandoeuvre-lès-Nancy, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Gardiennet
authors.affiliation: Univ. Grenoble Alpes, CEA,CNRS, F-, 38044, Grenoble, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Timmins
authors.affiliation: Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France. laurent.terradot@ibcp.fr.
authors.forename: Laurent
authors.initials: L
authors.lastname: Terradot
authors.affiliation: Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367, Lyon, France. a.bockmann@ibcp.fr.
authors.forename: Anja
authors.initials: A
authors.lastname: Böckmann
authors.affiliation: Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland. beme@ethz.ch.
authors.forename: Beat H
authors.identifier: 0000-0002-9107-4464
authors.initials: BH
authors.lastname: Meier
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000032:analysis / Q000378:metabolism*; D053488:DnaB Helicases / Q000737:chemistry / Q000378:metabolism*; D016480:Helicobacter pylori / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D004277:DNA, Single-Stranded; D053488:DnaB Helicases
keywords: helicase; magic-angle spinning; motor proteins; solid-state NMR spectroscopy