Rational design of ¹⁹F NMR labelling sites to probe protein structure and interactions

Proteins are investigated in increasingly more complex biological systems, where ¹⁹F NMR is proving highly advantageous due to its high gyromagnetic ratio and background-free spectra. Its application has, however, been hindered by limited chemical shift dispersions and an incomprehensive relationship between chemical shifts and protein structure. Here, we exploit the sensitivity of ¹⁹F chemical shifts to ring currents by designing labels with direct contact to a native or engineered aromatic ring. Fifty protein variants predicted by AlphaFold and molecular dynamics simulations show 80-90% success rates and direct correlations of their experimental chemical shifts with the magnitude of the engineered ring current. Our method consequently improves the chemical shift dispersion and through simple 1D experiments enables structural analyses of alternative conformational states, including ribosome-bound folding intermediates, and in-cell measurements of protein-protein interactions and thermodynamics. Our strategy thus provides a simple and sensitive tool to extract residue contact restraints from chemical shifts for previously intractable systems.