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Hyperpolarized water as universal sensitivity booster in biomolecular NMR

Abstract

NMR spectroscopy is the only method to access the structural dynamics of biomolecules at high (atomistic) resolution in their native solution state. However, this method’s low sensitivity has two important consequences: (i) typically experiments have to be performed at high concentrations that increase sensitivity but are not physiological, and (ii) signals have to be accumulated over long periods, complicating the determination of interaction kinetics on the order of seconds and impeding studies of unstable systems. Both limitations are of equal, fundamental relevance: non-native conditions are of limited pharmacological relevance, and the function of proteins, enzymes and nucleic acids often relies on their interaction kinetics. To overcome these limitations, we have developed applications that involve ‘hyperpolarized water’ to boost signal intensities in NMR of proteins and nucleic acids. The technique includes four stages: (i) preparation of the biomolecule in partially deuterated buffers, (ii) preparation of ‘hyperpolarized’ water featuring enhanced 1H NMR signals via cryogenic dynamic nuclear polarization, (iii) sudden melting of the cryogenic pellet and dissolution of the protein or nucleic acid in the hyperpolarized water (enabling spontaneous exchanges of protons between water and target) and (iv) recording signal-amplified NMR spectra targeting either labile 1H or neighboring 15N/13C nuclei in the biomolecule. Water in the ensuing experiments is used as a universal ‘hyperpolarization’ agent, rendering the approach versatile and applicable to any biomolecule possessing labile hydrogens. Thus, questions can be addressed, ranging from protein and RNA folding problems to resolving structure-function relationships of intrinsically disordered proteins to investigating membrane interactions.

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Fig. 1: Instrumental components underlying the dDNP methodology.
Fig. 2: The HyperW procedure.
Fig. 3: DNP of water.
Fig. 4: The dissolution-DNP experiment.
Fig. 5: NMR acquisition in hyperpolarized water experiments.
Fig. 6: The dissolution-DNP setup.
Fig. 7: Mapping of solvent exposure in IDPs through hyperpolarized water.
Fig. 8: Folding processes assessed by hyperpolarized water.
Fig. 9: Mapping of solvent-exposed surfaces.

Data availability

Data shown in Supplementary Fig. 4 can be found at https://zenodo.org/record/5774664#.YjfH6BDMKqA. All other data are published elsewhere and can be found as referenced in the main text.

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Acknowledgements

L.F. thanks G. Olsen, O. Szekely, M. Novakovic, K. Singh and C. Bretschneider, who contributed to his training and understanding of events involved in HyperW NMR. Research at the Weizmann Institute of Science is supported by the German-Israel Foundation (grant G-1501-302), the EU Horizon 2020 program (FET-OPEN grant 828946, PATHOS), Israel Science Foundation grant 965/18 and the Perlman Family Foundation. L.F. holds the Bertha and Isadore Gudelsky Professorial Chair and heads the Clore Institute for High-Field Magnetic Resonance Imaging and Spectroscopy, whose support is also acknowledged. C.H. acknowledges support from the National Institutes of Health (grant R01GM132655), the National Science Foundation (grant CHE-1362691) and the Welch Foundation (grant A-1658). D.K. acknowledges contributions from E. Canet, P. Kadeřávek, G. Olsen, D. Guarin and E. M. M. Weber and thanks G. Bodenhausen, F. Ferrage and D. Abergel for their support. The project leading to this application at the University of Vienna received funding from the European Research Council under the EU Horizon 2020 research and innovation programme (grant agreement 801936). Furthermore, this project was supported by the Austrian FWF (standalone grant no. P-33338).

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D.K. initiated and organized the collaboration leading to this Article. All authors wrote and approved the manuscript.

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Correspondence to Christian Hilty, Dennis Kurzbach or Lucio Frydman.

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Key references using this protocol

Novakovic, M. et al. Proc. Natl. Acad. Sci. USA 117, 2449–2455 (2020): https://www.pnas.org/content/117/5/2449

Kim, J. et al. J. Phys. Chem. Lett. 10, 5463–5467 (2019): https://doi.org/10.1021/acs.jpclett.9b02197

Szekely, O. et al. J. Am. Chem. Soc. 142, 9267–9284 (2020): https://doi.org/10.1021/jacs.0c00807

Kurzbach, D. et al. Angew. Chem. Int. Ed. Engl. 56, 389–392 (2017): https://doi.org/10.1002/anie.201608903

Sadet, A. et al. J. Am. Chem. Soc. 141, 12448–12452 (2019): https://doi.org/10.1021/jacs.9b03651

Kadeřávek, P. et al. Chemistry 24, 13418–13423 (2018): https://doi.org/10.1002/chem.201802885

Key data used in this protocol

Novakovic, M. et al. Proc. Natl. Acad. Sci. USA 117, 2449–2455 (2020): https://www.pnas.org/content/117/5/2449

Kim, J. et al. J. Magn. Reson. 326, 106942 (2021): https://doi.org/10.1016/j.jmr.2021.106942

Szekely, O. et al. J. Am. Chem. Soc. 142, 9267–9284 (2020): https://doi.org/10.1021/jacs.0c00807

Kurzbach, D. et al. Angew. Chem. Int. Ed. Engl. 56, 389–392 (2017): https://doi.org/10.1002/anie.201608903

Sadet, A. et al. J. Am. Chem. Soc. 141, 12448–12452 (2019): https://doi.org/10.1021/jacs.9b03651

Kadeřávek, P. et al. Chemistry 24, 13418–13423 (2018): https://doi.org/10.1002/chem.201802885

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Hilty, C., Kurzbach, D. & Frydman, L. Hyperpolarized water as universal sensitivity booster in biomolecular NMR. Nat Protoc (2022). https://doi.org/10.1038/s41596-022-00693-8

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