Biomolecular interactions studied by low-field NMR using SABRE hyperpolarization.

We demonstrate that low-field nuclear magnetic resonance provides a means for measuring biomacromolecular interactions without requiring a superconducting, or even a permanent magnet. A small molecule, 5-fluoropyridine-3-carboximidamide, is designed to be a specific ligand for the trypsin protein, while containing a fluorine atom as a nuclear spin hyperpolarizable label. With hyperpolarization by the parahydrogen based signal amplification by the reversible exchange method, fluorine NMR signals are detectable in the measurement field of 0.85 mT of an electromagnet, at a concentration of less than 100 μM. As a weak ligand for the protein, the hyperpolarized molecule can serve as a reporter for measuring the binding of other ligands of interest, illustrated by the determination of the dissociation constant of benzamidine from changes in the observed relaxation rates. A signal enhancement of more than 10 compared to Boltzmann polarization at the measurement field indicates that this experiment is not feasible without prepolarization. The extended magnetic field range for the measurement of biomolecular interactions under near physiological conditions, with a protein concentration on the order of 10 μM or less, provides a new option for screening of ligand binding, measurement of protein-protein interactions, and measurement of molecular dynamics.