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Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz

Nature Protocols volume 19, pages 406–440 (2024)Cite this article

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Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.

Key points

  • 28.2 T nuclear magnetic resonance spectrometers are now available and, thanks to their improved resolution, are especially useful for analyzing proteins that have flexible regions.

  • At such high magnetic fields, there are important challenges relating to the concomitant increase in spectral width. Key points explored in this protocol include the relaxation properties of proteins, choice of pulses for excitation and decoupling and setup of two-dimensional and multiple receiver experiments.

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Fig. 1: Schematic representation of the main topics discussed in this protocol.
Fig. 2: 2D CON spectra acquired at different magnetic fields (B0).
Fig. 3: 2D CACO spectra acquired at different magnetic fields (B0).
Fig. 4: A schematic representation of 2D 13C-detected experiments.
Fig. 5: A schematic representation of multiple receivers experiments.
Fig. 6: 1D 13C-detected NMR experiments acquired on α-synuclein.
Fig. 7: Three different kind of NMR tubes and respective spinners.
Fig. 8: Complementarity of different CON-based variants, acquired on CBP-ID4 at 283 K.
Fig. 9: CON and Hα flip CON comparison acquired on CBP-ID4 at 283 K.
Fig. 10: The mr_CON//HN Multiple Receivers experiment.
Fig. 11: An exclusively heteronuclear detected multiple receiver experiment: the mr_CON//Hα CAN.
Fig. 12: Complementarity of different CON-based variants, acquired on the BRCA1 construct at 308 K.
Fig. 13: Hα CACO experiment acquired on the BRCA1 construct at 308 K.

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Data availability

The data are available upon request to the authors.

Code availability

Pulse sequences are deposited at https://doi.org/10.6084/m9.figshare.23864817.

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Acknowledgements

This paper is part of a project funded by the European Union-NextGenerationEU through the ItaliaDomani PNRR project ‘Potentiating the Italian Capacity for Structural Biology Services in Instruct-ERIC’ (ITACA.SB, no. IR0000009). The support of the CERM/CIRMMP center of Instruct-ERIC and of the Italian Ministry for University and Research (MUR, FOE funding) is gratefully acknowledged. MUR and Bruker Switzerland AG are acknowledged for financial support to M.A.R. (DM 352/2022) and MUR for financial support to L.B. (Dipartimenti di Eccellenza 2018-2022). Further support has been provided by the ItaliaDomani PNRR projects ‘Tuscany Health Ecosystem’ (THE, no. ECS00000017) and ‘A multiscale integrated approach to the study of the nervous system in health and disease’ (MNESYS, no. PE0000006).

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Authors and Affiliations

  1. Department of Chemistry ‘Ugo Schiff’ and Magnetic Resonance Center (CERM), University of Florence, Florence, Italy

    Marco Schiavina, Lorenzo Bracaglia, Maria Anna Rodella, Isabella C. Felli & Roberta Pierattelli

  2. Bruker BioSpin AG, Fällanden, Switzerland

    Rainer Kümmerle

  3. Department of Computational and Structural Biology, Max Perutz Labs, University of Vienna, Vienna, Austria

    Robert Konrat

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  1. Marco Schiavina
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Contributions

M.S., I.C.F. and R.P. conceived and designed the protocol. All authors contributed to the NMR experiments. M.S., L.B. and M.A.R. analyzed the data. All authors wrote, read and commented on the paper.

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Correspondence to Marco Schiavina, Isabella C. Felli or Roberta Pierattelli.

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Key references using this protocol

Pontoriero, L. et al. Angew. Chem. Int. Ed. 59, 18537–18545 (2020): https://doi.org/10.1002/anie.202008079

Schiavina, M. et al. Biophys J. 117, 46–55 (2019): https://doi.org/10.1016/j.bpj.2019.05.017

Murrali, M. G. et al. Chembiochem. 19, 1625–1629 (2019) https://doi.org/10.1002/cbic.201800172

Banci, L. et al. GHz. Preprint at arXiv (2019): https://doi.org/10.48550/arXiv.1910.07462

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Schiavina, M., Bracaglia, L., Rodella, M.A. et al. Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz. Nat Protoc 19, 406–440 (2024). https://doi.org/10.1038/s41596-023-00921-9

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