Photo-CIDNP-Enhanced 19F NMR for Rapid Protein Conformation and Ligand Binding Analysis.

Abstract

19F NMR spectroscopy is a powerful probe of protein conformation and dynamics but is intrinsically limited by low sensitivity common to other NMR techniques. Here, we integrate photochemically induced dynamic nuclear polarization (photo-CIDNP) with genetically encoded 19F labels to enhance sensitivity by up to 220-fold, enabling the detection of 100 nM protein within 6.5 min. This approach employs commercially available, cost-effective 19F-labeled amino acid analogues and is broadly applicable to proteins ranging from 6.6 to 54.2 kDa. It allows rapid analysis of conformational changes and ligand binding in the low-micromolar range within minutes. Notably, it can readily characterize interactions with dissociation constants below 5 μM, which are challenging to access using conventional NMR. The combination of photo-CIDNP-induced sensitivity enhancement with the environmental responsiveness of 19F NMR establishes a practical platform for protein structural and interaction studies.