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Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning

Nature Protocols volume 12, pages 764782 (2017) | Download Citation

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Abstract

Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions—for example, in a lipid bilayer and at room temperature (0–35 °C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, 1H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional 13C-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

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Change history

  • 27 March 2017

    In the supplementary information originally posted online, the file: Supplementary Data 1–10 was not attached. The error has been corrected in the HTML and PDF versions as of 27 March 2017.

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Acknowledgements

We thank S. Lange and E. Ousby for valuable discussions. This work was supported by the Leibniz-Institut für Molekulare Pharmakologie, the Max Planck Society, the European Research Council (ERC Starting Grant to A.L.), the German Research Foundation (Deutsche Forschungsgemeinschaft; Emmy Noether Fellowship to A.L.) and the Fonds der Chemischen Industrie (Kekulé Scholarship to P.F.).

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Affiliations

  1. Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.

    • Pascal Fricke
    • , Veniamin Chevelkov
    • , Maximilian Zinke
    •  & Adam Lange
  2. Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

    • Karin Giller
    •  & Stefan Becker
  3. Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany.

    • Adam Lange

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Contributions

P.F., V.C. and M.Z. implemented the protocol; K.G. and S.B. produced the protein sample; V.C. and A.L. designed the research; P.F. and A.L. wrote the manuscript; and all authors commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Veniamin Chevelkov.

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https://doi.org/10.1038/nprot.2016.190

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