Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

CEITEC Central European Institute of Technology Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Chemical and Biological Physics Weizmann Institute of Science Rehovot Israel

Department of Structural and Computational Biology University of Vienna Vienna BioCenter 5 1030 Vienna Austria

Faculty of Chemistry Institute for Biological Chemistry University of Vienna Währinger Straße 38 1090 Vienna Austria

Laboratoire des biomolécules LBM Département de chimie École normale supérieure PSL University Sorbonne Université CNRS 75005 Paris France

Magnetic Resonance Center and Department of Chemistry Ugo Schiff University of Florence Via L Sacconi 6 50019 Sesto Fiorentino FI Italy

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Bah A, Forman-Kay JD. Modulation of intrinsically disordered protein function by post-translational modifications. J Biol Chem. 2016;291:6696–6705. doi: 10.1074/jbc.R115.695056. PubMed DOI PMC

Bertini I, Felli IC, Gonnelli L, Kumar MVV, Pierattelli R. 13C direct-detection biomolecular NMR spectroscopy in living cells. Angew Chem Int Ed Engl. 2011;50:2339–2341. doi: 10.1002/anie.201006636. PubMed DOI

Clemente N, et al. Osteopontin bridging innate and adaptive immunity in autoimmune diseases. J Immunol Res. 2016;2016:7675437. doi: 10.1155/2016/7675437. PubMed DOI PMC

Delaglio F, et al. NMRpipe—a multidimensional spectral processing system based on Unix pipes. J Biomol NMR. 1995;6:277–293. doi: 10.1007/BF00197809. PubMed DOI

Doll A, Bordignon E, Joseph B, Tschaggelar R, Jeschke G. Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures. J Magn Reson. 2012;222:34–43. doi: 10.1016/j.jmr.2012.06.003. PubMed DOI

Emsley L, Bodenhausen G. Gaussian pulse cascades: new analytical functions for rectangular selective inversion and in-phase excitation in NMR. Chem Phys Lett. 1990;165(2):469–475. doi: 10.1016/0009-2614(90)87025-M. DOI

Geen H. Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson. 1991;93:93–141.

Gil S, et al. NMR spectroscopic studies of intrinsically disordered proteins at near-physiological conditions. Angew Chem Int Ed. 2013;52:11808–11812. doi: 10.1002/anie.201304272. PubMed DOI

Goddard TD, Kneller DG. SPARKY. San Francisco: University of California; 2008.

Hamilton KS, Ellison MJ, Shaw GS. Identification of the ubiquitin interfacial residues in a ubiquitin–E2 covalent complex. J Biomol NMR. 2000;18:319–327. doi: 10.1023/A:1026773008237. PubMed DOI

Hyberts SG, Milbradt AG, Wagner AB, Arthanari H, Wagner G. Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. J Biomol NMR. 2012;52:315–327. doi: 10.1007/s10858-012-9611-z. PubMed DOI PMC

Kadeřávek P, Ferrage F, Bodenhausen G, Kurzbach D. High-resolution NMR of folded proteins in hyperpolarized physiological solvents. Chem Eur J. 2018;24:13418–13423. doi: 10.1002/chem.201802885. PubMed DOI

Katsikis S, Marin-Montesinos I, Pons M, Ludwig C, Gunther UL. Improved stability and spectral quality in ex situ dissolution DNP using an improved transfer device. Appl Magn Reson. 2015;46:723–729. doi: 10.1007/s00723-015-0680-5. DOI

Kazimierczuk K, Zawadzka A, Kozminski W. Narrow peaks and high dimensionalities: exploiting the advantages of random sampling. J Magn Reson. 2009;197:219–228. doi: 10.1016/j.jmr.2009.01.003. PubMed DOI

Kazimierczuk K, Zawadzka-Kazimierczuk A, Kozminski W. Non-uniform frequency domain for optimal exploitation of non-uniform sampling. J Magn Reson. 2010;205:286–292. doi: 10.1016/j.jmr.2010.05.012. PubMed DOI

Kim J, Liu M, Hilty C. Modeling of polarization transfer kinetics in protein hydration using hyperpolarized water. J Phys Chem B. 2017;121:6492–6498. doi: 10.1021/acs.jpcb.7b03052. PubMed DOI

Kupce E, Freeman R. Wideband excitation with polychromatic pulses. J Magn Reson A. 1994;108:268–273. doi: 10.1006/jmra.1994.1123. DOI

Kurzbach D, et al. Cooperative unfolding of compact conformations of the intrinsically disordered protein osteopontin. Biochemistry. 2013;52:5167–5175. doi: 10.1021/bi400502c. PubMed DOI PMC

Kurzbach D, et al. Investigation of intrinsically disordered proteins through exchange with hyperpolarized water. Angew Chem Int Ed Engl. 2017;56:389–392. doi: 10.1002/anie.201608903. PubMed DOI

Lescop E, Schanda P, Brutscher B. A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J Magn Reson. 2007;187:163–169. doi: 10.1016/j.jmr.2007.04.002. PubMed DOI

Lipso KW, Bowen S, Rybalko O, Ardenkjaer-Larsen JH. Large dose hyperpolarized water with dissolution-DNP at high magnetic field. J Magn Reson. 2017;274:65–72. doi: 10.1016/j.jmr.2016.11.008. PubMed DOI

Markley JL, et al. Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy. J Biomol NMR. 1998;12:1–23. doi: 10.1023/A:1008290618449. PubMed DOI

Mateos B, Konrat R, Pierattelli R, Felli IC. NMR characterization of long-range contacts in intrinsically disordered proteins from paramagnetic relaxation enhancement in 13C direct-detection experiments. ChemBioChem. 2019;20:335–339. doi: 10.1002/cbic.201800539. PubMed DOI

Mayzel M, Rosenlow J, Isaksson L, Orekhov VY. Time-resolved multidimensional NMR with non-uniform sampling. J Biomol NMR. 2014;58:129–139. doi: 10.1007/s10858-013-9811-1. PubMed DOI PMC

Milani J, et al. A magnetic tunnel to shelter hyperpolarized fluids. Rev Sci Instrum. 2015;86:024101. doi: 10.1063/1.4908196. PubMed DOI

Modig K, Liepinsh E, Otting G, Halle B. Dynamics of protein and peptide hydration. J Am Chem Soc. 2004;126:102–114. doi: 10.1021/ja038325d. PubMed DOI

Mori S, Abeygunawardana C, Johnson MO, Zijl PCV. Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new Fast HSQC (FHSQC) detection scheme that avoids water saturation. J Magn Reson B. 1995;108:94–98. doi: 10.1006/jmrb.1995.1109. PubMed DOI

Nucci NV, Pometun MS, Wand AJ. Site-resolved measurement of water–protein interactions by solution NMR. Nat Struct Mol Biol. 2011;18:245–249. doi: 10.1038/nsmb.1955. PubMed DOI PMC

Olsen G, Markhasin E, Szekely O, Bretschneider C, Frydman L. Optimizing water hyperpolarization and dissolution for sensitivity-enhanced 2D biomolecular NMR. J Magn Reson. 2016;264:49–58. doi: 10.1016/j.jmr.2016.01.005. PubMed DOI

Otting G. NMR studies of water bound to biological molecules. Prog Nucl Magn Reson Spectrosc. 1997;31:259–285. doi: 10.1016/S0079-6565(97)00012-5. DOI

Platzer G, et al. The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites. Biochemistry. 2011;50:6113–6124. doi: 10.1021/bi200291e. PubMed DOI

Ragavan M, Iconaru LI, Park CG, Kriwacki RW, Hilty C. Real-time analysis of folding upon binding of a disordered protein by using dissolution DNP NMR spectroscopy. Angew Chem Int Ed Engl. 2017;56:7070–7073. doi: 10.1002/anie.201700464. PubMed DOI PMC

Reichheld SE, Muiznieks LD, Keeley FW, Sharpe S. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc Natl Acad Sci U S A. 2017;114(22):E4408–E4415. doi: 10.1073/pnas.1701877114. PubMed DOI PMC

Rodrigues LR, Teixeira JA, Schmitt FL, Paulsson M, Lindmark-Mansson H. The role of osteopontin in tumor progression and metastasis in breast cancer. Cancer Epidemiol Biomark Prev. 2007;16:1087–1097. doi: 10.1158/1055-9965.EPI-06-1008. PubMed DOI

Schanda P, Kupce E, Brutscher B. SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR. 2005;33:199–211. doi: 10.1007/s10858-005-4425-x. PubMed DOI

Smith MA, Hu H, Shaka AJ. Improved broadband inversion performance for NMR in liquids. J Magn Reson. 2001;151:269–283. doi: 10.1006/jmre.2001.2364. DOI

Szekely O, Olsen GL, Felli IC, Frydman L. High-resolution 2D NMR of disordered proteins enhanced by hyperpolarized water. Anal Chem. 2018;90(10):6169–6177. doi: 10.1021/acs.analchem.8b00585. PubMed DOI

Theillet FX, et al. Structural disorder of monomeric alpha-synuclein persists in mammalian cells. Nature. 2016;530:45–50. doi: 10.1038/nature16531. PubMed DOI

Viennet T, et al. Selective protein hyperpolarization in cell lysates using targeted dynamic nuclear polarization. Angew Chem Int Ed Engl. 2016;55:10746–10750. doi: 10.1002/anie.201603205. PubMed DOI

Vijay-Kumar S, Bugg C, Cook WJ. Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol. 1987;194:531–544. doi: 10.1016/0022-2836(87)90679-6. PubMed DOI

Wang Y, Hilty C. Determination of ligand binding epitope structures using polarization transfer from hyperpolarized ligands. J Med Chem. 2019;62:2419–2427. doi: 10.1021/acs.jmedchem.8b01711. PubMed DOI

Yuwen T, Skrynnikov NR. CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions. J Biomol NMR. 2014;58:175–192. doi: 10.1007/s10858-014-9815-5. PubMed DOI

Zawadzka-Kazimierczuk A, Kozminski W, Sanderova H, Krasny L. High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins. J Biomol NMR. 2012;52:329–337. doi: 10.1007/s10858-012-9613-x. PubMed DOI PMC

Toward protein NMR at physiological concentrations by hyperpolarized water-Finding and mapping uncharted conformational spaces