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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. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS QUANTIFYING THE THERMODYNAMICS OF PROTEIN UNFOLDING USING

2D NMR SPECTROSCOPY Article Open access 07 August 2020 CHARACTERIZING PROTEINS IN A NATIVE BACTERIAL ENVIRONMENT USING SOLID-STATE NMR SPECTROSCOPY Article 13 January 2021 ENSEMBLE

DETERMINATION BY NMR DATA DECONVOLUTION Article 11 May 2023 DATA AVAILABILITY The data are available upon request to the authors. CODE AVAILABILITY Pulse sequences are deposited at

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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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 * Bruker BioSpin AG, Fällanden, Switzerland Rainer

Kümmerle * Department of Computational and Structural Biology, Max Perutz Labs, University of Vienna, Vienna, Austria Robert Konrat Authors * Marco Schiavina View author publications You

can also search for this author inPubMed Google Scholar * Lorenzo Bracaglia View author publications You can also search for this author inPubMed Google Scholar * Maria Anna Rodella View

author publications You can also search for this author inPubMed Google Scholar * Rainer Kümmerle View author publications You can also search for this author inPubMed Google Scholar *

Robert Konrat View author publications You can also search for this author inPubMed Google Scholar * Isabella C. Felli View author publications You can also search for this author inPubMed 

Google Scholar * Roberta Pierattelli View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHORS Correspondence to

Marco Schiavina, Isabella C. Felli or Roberta Pierattelli. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature

Protocols_ thanks Davy Sinnaeve and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS 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 SUPPLEMENTARY

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