The structure of a ligand bound to a protein is determined from fast pseudo-3D NMR spectroscopy with transfer of hyperpolarization.

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R 2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.