For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13C/15N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (1H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3–5 days.
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DNP-supported solid-state NMR studies of 13C,15N,29Si-enriched biosilica of Cyclotella cryptica and Thalassiosira pseudonana
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Source data are provided with this paper. All other data supporting the approach described in this protocol are available from the corresponding authors upon reasonable request.
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The authors thank H. van Ingen for providing access to the solution-state NMR instrument, and D. Mance and Dr. Klaartje Houben for technical support and discussions. We are indebted to P. Tordo and O. Ouari (Aix-Marseille Université) for providing AMUPol for the DNP experiments S.N. was supported by the Netherlands’ Magnetic Resonance Research School (NMARRS, project number 022.005.029). Furthermore, this work was supported by the Dutch Research Council (NWO, projects 700.26.121 and 700.10.443 to M.B.) and by iNEXT-Discovery (project number 871037), a project funded by the Horizon 2020 program of the European Commission.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Lukas Trantirek and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol:
Chordia, S. et al. Preprint at ChemRxiv (2020): https://doi.org/10.26434/chemrxiv.12485993.v1
Pinto, C. et al. J Struct Biol 206, 1−11 (2019): https://doi.org/10.1016/j.jsb.2017.11.015.
Baker, L. A. et al. Structure 26, 161−170 (2018): https://doi.org/10.1016/j.str.2017.11.011
Baker, L. A. et al. J Biomol NMR 62, 199−208 (2015): https://doi.org/10.1007/s10858-015-9936-5
Supplementary Figs. 1–9, Supplementary Table 1 and Supplementary Notes 1–7.
Biorad format .1sc unprocessed gel image, where the last four lanes correspond to Supplementary Fig. 2
1D Bruker-format unprocessed spectra.
2D Bruker-format unprocessed spectra.
2D Bruker-format unprocessed spectra.
1D Bruker-format unprocessed spectra.
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Narasimhan, S., Pinto, C., Lucini Paioni, A. et al. Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy. Nat Protoc 16, 893–918 (2021). https://doi.org/10.1038/s41596-020-00439-4