Hyperpolarizing Proteins by Exchange (“HYPEX”)

Phantom IRM

(Left) Hyperpolarized HN-CON correlation spectrum of osteopontin detected in 55 s using hyperpolarized HDO, in ca. 98% deuterated buffer, with assignments of a few well-resolved residues. (Right) conventional reference spectrum acquired in 10% deuterated buffer

Signal enhancements of up to two orders of magnitude 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. Hyperpolarized 3D spectroscopy opens the possibility of D-DNP studies of larger proteins and IDPs. The signal enhancements obtained depend 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 makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues in acquisition times of just over a minute. The proposed experiments have been applied to the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).




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