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

Area of Science:

• Biophysics
• Chemical Biology
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Nuclear spin-lattice relaxation rate (1/T1) is influenced by molecular correlation times (τc).
• Detecting changes in τc upon ligand-protein binding is challenging at high magnetic fields due to limited sensitivity.
• Existing methods struggle to provide significant relaxation rate differences for probing binding events.

Purpose of the Study:

• To develop a sensitive NMR-based scheme for detecting protein-ligand interactions.
• To enhance the contrast in relaxation rates for improved detection sensitivity.
• To enable reliable detection of protein binding at low concentrations and with minimal labeled compounds.

Main Methods:

• Utilizing dynamic nuclear polarization (DNP) for hyperpolarization of nuclear spins.
• Implementing low magnetic field relaxation to generate substantial T1 contrast.
• Employing high-field liquid-state NMR for readout of low-field relaxation effects.
• Developing competitive binding assays for label-free ligand detection.

Main Results:

• Achieved reliable detection of protein binding using only 14 μM of a 13C-labeled reporter ligand.
• Demonstrated reliable observation of protein binding at protein concentrations as low as 2 μM in a single scan.
• Successfully expanded the scheme for label-free ligand detection via competitive binding experiments.

Conclusions:

• The proposed DNP-enhanced, low-field relaxation NMR scheme significantly improves sensitivity for detecting protein-ligand interactions.
• This method offers a powerful tool for studying biomolecular interactions at low concentrations and with minimal labeling.
• The technique is adaptable for both labeled and label-free ligand binding studies.