Amplification of Nuclear Overhauser Effect Signals by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins
- 8 September 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 92 (20), 13718-13723
- https://doi.org/10.1021/acs.analchem.0c01071
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.Keywords
Funding Information
- Division of Chemistry (CHE-1362691)
- Welch Foundation (A-1658)
- Texas A and M University
This publication has 33 references indexed in Scilit:
- Investigating Protein–Ligand Interactions by Solution Nuclear Magnetic Resonance SpectroscopyChemphyschem, 2018
- Fragment-based drug discovery using NMR spectroscopyJournal of Biomolecular NMR, 2013
- NMR-based analysis of protein–ligand interactionsAnalytical and Bioanalytical Chemistry, 2013
- Fragment Screening of GPCRs Using Biophysical Methods: Identification of Ligands of the Adenosine A2A Receptor with Novel Biological ActivityACS Chemical Biology, 2012
- Fragment Screening of Stabilized G-Protein-Coupled Receptors Using Biophysical MethodsMethods in Enzymology, 2011
- Perspectives on NMR in drug discovery: a technique comes of ageNature Reviews Drug Discovery, 2008
- TINS, Target Immobilized NMR Screening: An Efficient and Sensitive Method for Ligand DiscoveryCell Chemical Biology, 2005
- Exchange-transferred NOE spectroscopy and bound ligand structure determinationCurrent Opinion in Structural Biology, 2003
- NMR Spectroscopy Techniques for Screening and Identifying Ligand Binding to Protein ReceptorsAngewandte Chemie, 2003
- Recent developments in transferred NOE methodsProgress in Nuclear Magnetic Resonance Spectroscopy, 1994