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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 1 H- 15 N 2D correlation experiments. Here we introduce 2D 13 C-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).

Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis-associated intrinsically disordered protein (IDP), at near-physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solvent-exposed residues and therefore selectively enhances signals in 1 H- 15 N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.

Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis-associated intrinsically disordered protein (IDP), at near-physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solvent-exposed residues and therefore selectively enhances signals in 1H-15N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN. [ABSTRACT FROM AUTHOR]

Intrinsisch unstrukturierte Proteine (IUPs) spielen eine zentrale Rolle in Proteininteraktionsnetzwerken als wichtige Knotenpunkte und Kopplungsstellen. Hier zeigen wir mit einer Kombination aus NMR- und ESR-Spektroskopie, dass die Bindung des Cytokins Osteopontin an seinen natürlichen Liganden Heparin von thermodynamisch kompensierend wirkenden Strukturanpassungen begleitet wird: Das Kernsegment des OPN entfaltet sich, und dieses 'Entfalten durch Bindung' kompensiert entropische Verluste durch die Heparinbindung. Es wird gezeigt, dass dieser Prozess primär durch elektrostatische Wechselwirkungen zwischen Heparin und geladenen Regionen entlang des Proteinrückgrats bestimmt wird. Dieses Wechselspiel zwischen Struktur und Thermodynamik ist auch von grundlegender Bedeutung für IUPs zur schnellen Substratbindung und -freisetzung innerhalb von Wechselwirkungsnetzwerken. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This 'unfolding-upon-binding' is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin-OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate-binding and -release events in IDP interaction networks. [ABSTRACT FROM AUTHOR]

Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of “fuzzy complexes.” Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of “energetic frustration” and provides an explanation for the puzzling observation of disordered allostery in IDPs. [ABSTRACT FROM AUTHOR]