The protein-ligand binding affinity is evaluated using low-cost, low-field nuclear magnetic resonance (NMR) spectroscopy at 0.85 mT. Strong signals are achieved through the hyperpolarization of 1H nuclei by parahydrogen-based signal amplification by reversible exchange (SABRE). The interaction is monitored by tracking the ubiquitous hydrogen signal. Despite the hyperpolarization, a key challenge of the label-free detection at low field is the distinction of signals in the absence of chemical shift. 1H signals of ligands are measured after deuterating the coligand and solvent and numerically accounting for orthohydrogen signal, which is produced during the hyperpolarization process. Spin-spin (R2) relaxation rates quantify the protein-ligand interaction. Since R2 in a milli-Tesla field does not include an exchange contribution, the calculation of the ligand dissociation constant KD is simplified compared to traditional high-field NMR. The technique for monitoring 1H signals is generalizable to detect any ligand that is competitively binding to the protein. It expands the application of low-field NMR for high-throughput screening and for studies of biochemical processes that involve ligand interactions.
Roy et al. (Tue,) studied this question.