Abstract
Immobilization of a target protein enhances the cross-relaxation rates for transfer of nuclear spin polarization but reduces the accessible target concentration. Hyperpolarization of ligand spins by dissolution dynamic nuclear polarization (D-DNP) is shown to increase sensitivity for observing the intraligand nuclear Overhauser effect (NOE). This effect, also known as the transferred NOE (trNOE), can be used for detection of binding and for obtaining binding related structural information. The measurement of hyperpolarized trNOE signals is demonstrated for the ligand 4’-hydroxyazobenzene-2-carboxylic acid inter-acting with avidin protein immobilized on polystyrene beads. In a sample containing 63.5 μM ligand and 0.83 μM accessible protein binding site, the signal enhancement provided by D-DNP leads to single-scan detection of the NOE buildup, despite that this signal peaks at only 2% of the total ligand signal. These buildup curves allow the confirmation of binding through a change in the sign of the NOE, and the quantitative determination of cross-relaxation rates. The combination of the D-DNP technique and protein immobilization may facilitate the identification of intraligand NOEs in ligand screening for drug discovery. The same method may be applied to in vivo characterization of ligand interactions with cell surface proteins.
Funding Information
  • Division of Chemistry (CHE-1362691)
  • Welch Foundation (A-1658)
  • Texas A and M University