Insights into the Structure and Dynamics of Proteins from ¹⁹F Solution NMR Spectroscopy

¹⁹F NMR spectroscopy has recently witnessed a resurgence as an attractive analytical tool for the study of the structure and dynamics of biomolecules in vitro and in cells, despite reports of its applications in biomolecular NMR since the 1970s. The high gyromagnetic ratio, large chemical shift dispersion, and complete absence of the spin 1/2 ¹⁹F nucleus from biomolecules results in background-free, high-resolution ¹⁹F NMR spectra. The introduction of ¹⁹F probes in a few selected locations in biomolecules reduces spectral crowding despite its increased line width in comparison to typical ¹H NMR line widths and allows rapid site-specific measurements from simple 1D spectra alone. The design and synthesis of novel ¹⁹F probes with reduced line widths and increased chemical shift sensitivity to the surrounding environment, together with advances in labeling techniques, NMR methodology, and hardware, have overcome several drawbacks of ¹⁹F NMR spectroscopy. The increased interest and widespread use of ¹⁹F NMR spectroscopy of biomolecules is gradually establishing it as a sensitive and high-resolution probe of biomolecular structure and dynamics, supplementing traditional ¹³C/¹⁵N-based methods. This Review focuses on the advances in ¹⁹F solution NMR spectroscopy of proteins in the past 5 years, with an emphasis on novel ¹⁹F tags and labeling techniques, NMR experiments to probe protein structure and conformational dynamics in vitro, and in-cell NMR applications.