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

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

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

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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