Monitoring ssDNA Binding to the DnaB Helicase from <i>Helicobacter pylori</i> by Solid‐State NMR Spectroscopy

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  • Thomas Wiegand
    Physical Chemistry ETH Zurich 8093 Zurich Switzerland
  • Riccardo Cadalbert
    Physical Chemistry ETH Zurich 8093 Zurich Switzerland
  • Carole Gardiennet
    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
  • Joanna Timmins
    Univ. Grenoble Alpes CEA,CNRS, F- 38044 Grenoble France
  • Laurent Terradot
    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
  • Anja Böckmann
    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
  • Beat H. Meier
    Physical Chemistry ETH Zurich 8093 Zurich Switzerland

説明

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>Helicobacter pylori</jats:italic> using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. <jats:sup>13</jats:sup>C 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 <jats:italic>H. pylori</jats:italic> 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.</jats:p>

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