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

The nuclear spin-lattice relaxation rate 1/T₁ depends on the correlation time τ_c of the molecule bearing the nuclear spin, and can therefore probe changes of τ_c 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.