Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the blind sphere around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (similar to 14 angstrom) than for nitroxide (similar to 11 angstrom) in C-13-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly. (C) 2021 The Author(s). Published by Elsevier Inc.

Automated summary

Labeling biomolecules with a paramagnetic probe for NMR spectroscopy allows determining long-range distance restraints, aiding in the structure determination of large proteins and protein complexes. The success of site-directed labeling of bacterial DnaB helicase with maleimide tags was confirmed through EPR experiments, leading to the extraction of site-specific PREs for further characterization. Comparison of experimental PREs and computational modeling of nitroxide and Gd3+ tags revealed differences in the blind sphere size around the paramagnetic center.