Biomolecular interactions studied by low-field NMR using SABRE hyperpolarization

Area of Science:

• Biophysics
• Chemical Biology
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Traditional nuclear magnetic resonance (NMR) spectroscopy requires high magnetic fields, limiting its application in biomolecular interaction studies under physiological conditions.
• Measuring weak biomolecular interactions, such as ligand binding to proteins, often necessitates sensitive detection methods.
• Hyperpolarization techniques offer a significant signal enhancement for NMR, potentially enabling measurements at lower magnetic fields.

Purpose of the Study:

• To demonstrate the feasibility of measuring biomacromolecular interactions using low-field nuclear magnetic resonance (NMR).
• To develop a method utilizing a hyperpolarized fluorine-containing molecule as a reporter for ligand binding.
• To assess the potential of this technique for screening ligand binding and studying protein interactions under near-physiological conditions.

Main Methods:

• Design of a specific ligand (5-fluoropyridine-3-carboximidamide) for trypsin, containing a hyperpolarizable fluorine-19 nucleus.
• Utilized parahydrogen-based signal amplification by reversible exchange (PHABRE) for hyperpolarizing the fluorine label.
• Performed NMR measurements at a low magnetic field (0.85 mT) using an electromagnet, detecting signals at sub-100 μM concentrations.

Main Results:

• Achieved a signal enhancement exceeding 10^6-fold compared to Boltzmann polarization, making low-field NMR feasible.
• Successfully detected the binding of benzamidine to trypsin by monitoring changes in relaxation rates (R2) of the hyperpolarized ligand.
• Determined the dissociation constant (KD) for benzamidine-trypsin interaction, demonstrating the quantitative capability of the method.

Conclusions:

• Low-field NMR, enhanced by hyperpolarization, provides a sensitive and accessible platform for studying biomolecular interactions.
• This technique overcomes the limitations of superconducting magnets, enabling measurements under near-physiological conditions with low protein concentrations.
• The method offers a promising new avenue for drug discovery, protein-protein interaction studies, and molecular dynamics investigations.