Label-Free Measurement of Ligand Interactions Using SABRE Hyperpolarization at Low Magnetic Fields.

Area of Science:

• Biochemistry
• Spectroscopy
• Chemical Biology

Background:

• Protein-ligand interactions are crucial in biological processes.
• Traditional Nuclear Magnetic Resonance (NMR) spectroscopy for binding studies often requires high fields and can be costly.
• Label-free detection methods are desirable for efficient screening.

Purpose of the Study:

• To evaluate protein-ligand binding affinity using low-field NMR.
• To leverage hyperpolarization techniques for enhanced signal detection.
• To develop a simplified method for calculating ligand dissociation constants.

Main Methods:

• Utilized low-field (0.85 mT) NMR spectroscopy.
• Employed parahydrogen-based signal amplification by reversible exchange (SABRE) for ¹H nuclei hyperpolarization.
• Monitored ubiquitous hydrogen signals and quantified protein-ligand interaction using spin-spin (R₂) relaxation rates.

Main Results:

• Achieved strong signals for protein-ligand binding detection via hyperpolarization.
• Overcame challenges of label-free detection at low fields by deuterating coligand/solvent and accounting for orthohydrogen signals.
• Simplified K_D calculation due to the absence of exchange contribution to R₂ at milli-Tesla fields.

Conclusions:

• Developed a generalizable technique for monitoring ¹H signals in protein-ligand interactions.
• Demonstrated the utility of low-field NMR with SABRE for label-free, high-throughput screening.
• Expanded applications of NMR for studying biochemical processes involving ligand binding.