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bioRxiv · 10.1101/2021.09.14.460235

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

Zehnder, J.·Cadalbert, R.·Yulikov, M.·Kuenze, G.·Wiegand, T.
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Abstract

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+ (~14 [A]) than for nitroxide (~11 [A]) in 13C-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.

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Zehnder, J., Cadalbert, R., Yulikov, M., Kuenze, G., Wiegand, T.. 2021-09-14. Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR. https://doi.org/10.1101/2021.09.14.460235

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