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 & mu;M. 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 KD of benzamidine from changes in the observed R2 relaxation rates. A signal enhancement of more than 106 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 & mu;M or less, provides a new option for screening of ligand binding, measurement of protein-protein interactions, and measurement of molecular dynamics. Nuclear spin hyperpolarization by parahydrogen enables the measurement of biomolecular interactions without the need for a superconducting or permanent magnet. Observed is a fluorine signal of a purpose-designed reporter ligand for a target protein.

Automated summary

Low-field nuclear magnetic resonance enables the measurement of biomolecular interactions without the need for a superconducting or permanent magnet. A small molecule with a hyperpolarized fluorine label is designed as a specific ligand for a target protein, and its nuclear magnetic resonance signals can be detected at a concentration of less than 100 μM. This technique allows for the measurement of ligand binding and protein-protein interactions.