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Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa motor protein: identification of structural variability

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Abstract

Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural “hotspots” for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.

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Acknowledgements

This work was supported by the Swiss National Science Foundation (Grant 200020_159707), and the ETH Career SEED-69 16-1. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No. 741863, FASTER).

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Authors and Affiliations

  1. Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland

    Thomas Wiegand, Riccardo Cadalbert & Beat H. Meier

  2. Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland

    Christine von Schroetter & Frédéric H.-T. Allain

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  1. Thomas Wiegand
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  2. Riccardo Cadalbert
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  3. Christine von Schroetter
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  4. Frédéric H.-T. Allain
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  5. Beat H. Meier
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Corresponding authors

Correspondence to Frédéric H.-T. Allain or Beat H. Meier.

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Wiegand, T., Cadalbert, R., von Schroetter, C. et al. Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa motor protein: identification of structural variability. J Biomol NMR 71, 237–245 (2018). https://doi.org/10.1007/s10858-018-0196-z

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  • DOI: https://doi.org/10.1007/s10858-018-0196-z

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