Residue-resolved monitoring of protein hyperpolarization at sub-second time resolution

Signal-enhancement techniques for NMR spectroscopy are important to amplify the weak resonances provided by nuclear spins. Recently, 'hyperpolarization' techniques have been intensively investigated. These provide nuclear spin states far from equilibrium yielding strong signal boosts up to four orders of magnitude. Here we propose a method for real-time NMR of 'hyperpolarized' proteins at residue resolution. The approach is based on dissolution dynamic nuclear polarization (d-DNP), which enables the use of hyperpolarized buffers that selectively boost NMR signals of solvent-exposed protein residues. The resulting spectral sparseness and signal enhancements enable recording of residue-resolved spectra at a 2 Hz sampling rate. Thus, we monitor the hyperpolarization level of different protein residues simultaneously under near-physiological conditions. We aim to address two points: 1) NMR experiments are often performed under conditions that increase sensitivity but are physiologically irrelevant; 2) long signal accumulation impedes fast real-time monitoring. Both limitations are of fundamental relevance to ascertain pharmacological relevance and study protein kinetics. Dissolution dynamic nuclear polarisation allows for dramatic signal enhancement in protein NMR spectroscopy, but loss of polarisation limits temporal and structural resolution. Here polarisation of the solvent and subsequent transfer to the target molecule enables selective detection of hyperpolarised residues.

Automated summary

A method for real-time NMR of 'hyperpolarized' proteins at residue resolution is proposed using dissolution dynamic nuclear polarization. This approach enables selective detection of hyperpolarized residues, addressing limitations in sensitivity and monitoring speed in protein NMR spectroscopy. Hyperpolarization of solvent and subsequent transfer to target molecules significantly enhances signal in protein NMR, allowing for residue-resolved spectra recording at a high sampling rate.