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

We propose a method for real-time nuclear magnetic resonance (NMR) spectroscopy 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 backbone amides that incur magnetization fast from their surroundings. Capitalizing on the resulting spectral sparseness and simultaneous signal enhancement, we obtained residue-resolved NMR spectra at a sampling rate of 2 Hz. We could thus track the evolution of hyperpolarization at different protein residues simultaneously with time. This was achieved under near-physiological conditions, i.e., in aqueous solution at physiological salt concentration and at 37° C. With this development, two often encountered limitations of conventional solution-state NMR can be addressed: 1) NMR experiments are typically performed under conditions that increase sensitivity but are physiologically not relevant (low pH, low temperature) and; 2) signal accumulation over long periods impedes the determination of fast (on the order of seconds) real-time monitoring. Both limitations are of equal fundamental relevance: interaction studies under non-native conditions are of limited pharmacological relevance, and the key to the function of proteins often resides in their interaction kinetics. The proposed technique possibly opens new routes towards residue and temporally resolved spectroscopy at the atomistic level by overcoming the need for signal averaging in residue-resolved protein biomolecular NMR.