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ATPase Site Configuration of the RNA Helicase DbpA Probed by ENDOR Spectroscopy

  • Ilia Kaminker
  • Daniella GoldfarbEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

Electron-nuclear double resonance (ENDOR) is a method that probes the local structure of paramagnetic centers via their hyperfine interactions with nearby magnetic nuclei. Here we describe the use of this technique to structurally characterize the ATPase active site of the RNA helicase DbpA, where Mg2+-ATP binds. This is achieved by substituting the EPR (electron paramagnetic resonance) silent Mg2+ ion with paramagnetic, EPR active, Mn2+ ion. 31P ENDOR provides the interaction of the Mn2+ with the nucleotide (ADP, ATP and its analogs) through the phosphates. The ENDOR spectra clearly distinguish between ATP- and ADP-binding modes. In addition, by preparing 13C-enriched DbpA, 13C ENDOR is used to probe the interaction of the Mn2+ with protein residues. This combination allows tracking structural changes in the Mn2+ coordination shell, in the ATPase site, in different states of the protein, namely with and without RNA and with different ATP analogs. Here, a detailed description of sample preparation and the ENDOR measurement methodology is provided, focusing on measurements at W-band (95 GHz) where sensitivity is high and spectral interpretations are relatively simple.

Key words

RNA helicase DbpA ATPase Mn2+ ATP ADP AMPPnP EPR ENDOR W-band EPR 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Chemical PhysicsWeizmann Institute of ScienceRehovotIsrael

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