ATPase site configuration of the RNA helicase DbpA probed by ENDOR spectroscopy.

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 Mg(2+)-ATP binds. This is achieved by substituting the EPR (electron paramagnetic resonance) silent Mg(2+) ion with paramagnetic, EPR active, Mn(2+) ion. (31)P ENDOR provides the interaction of the Mn(2+) 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 (13)C-enriched DbpA, (13)C ENDOR is used to probe the interaction of the Mn(2+) with protein residues. This combination allows tracking structural changes in the Mn(2+) 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.