# Single-scan detection of ligand-binding using hyperpolarization and low-field relaxation

## TL;DR

This paper introduces a new method using hyperpolarization and low-field relaxation to detect ligand binding to proteins with high sensitivity and low sample concentrations.

## Contribution

The novel approach uses low-field relaxation with hyperpolarization to achieve high T1 contrast for ligand-binding detection.

## Key findings

- Protein binding was reliably observed at concentrations as low as 2 μM using 14 μM of a 13C-labeled reporter ligand.
- The method was expanded to detect label-free ligands through competitive binding experiments.
- Low-field relaxation provides a large T1 contrast, enabling sensitive ligand-binding detection.

## Abstract

The nuclear spin-lattice relaxation rate 1/T1 depends on the correlation time τc of the molecule bearing the nuclear spin, and can therefore probe changes of τc upon binding of a rapidly moving small ligand to a more slowly moving larger protein. In practice however, the dependence is such that only a small difference in relaxation rate is obtained at high field. Here we present a scheme in which nuclear spins are first hyperpolarized using DNP, and then allowed to relax at low magnetic field in presence of a target protein, which generates a large T1 contrast. The sample is subsequently transferred into a conventional nuclear magnetic resonance probe (NMR), where the effect of the low-field relaxation is read out using high-field liquid-state NMR. Using only 14 μM of a 13C-labeled reporter ligand, we observe protein binding reliably for protein concentrations as low as 2 μM in a single scan. The scheme is expanded to a label-free ligand via a competitive binding experiment in which the label-free ligand displaces the 13C-labeled reporter ligand.

The nuclear spin-lattice relaxation rate can be used to probe binding of a rapidly moving small ligand to a more slowly moving larger protein, however, the measurements are challenging at high magnetic fields. Here, the authors report low-field relaxation as a sensitive contrast mechanism for detecting ligand binding, using a hyperpolarization experiment to detect ligand signals with high repeatability at concentrations as low as 14 μM.