Label-Free Measurement of Ligand Interactions Using SABRE Hyperpolarization at Low Magnetic Fields

TL;DR

This paper introduces a low-cost, low-field NMR method using SABRE hyperpolarization to measure protein-ligand binding affinities without labeling.

Contribution

A novel label-free technique for ligand interaction measurement using low-field NMR and SABRE hyperpolarization.

Findings

1H signals of ligands can be measured by deuteration and accounting for orthohydrogen.

Spin–spin relaxation rates (R2) simplify K_D calculations in low magnetic fields.

The method is generalizable for high-throughput screening and biochemical process studies.

Abstract

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 (R
2) relaxation rates quantify the protein–ligand interaction.
Since R
2 in a milli-Tesla field does not
include an exchange contribution, the calculation of the ligand dissociation
constant K
D 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.

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