Integrating 19F Distance Restraints for Accurate Protein Structure Determination by Magic Angle Spinning NMR Spectroscopy

Traditional protein structure determination by magic angle spinning (MAS) solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from ¹³C-, ¹⁵N-, and ¹H-based dipolar-based correlation experiments. Here, we show that ¹⁹F fast (60 kHz) MAS NMR spectroscopy can supply additional, longer distances. Using 4F-Trp,U-¹³C,¹⁵N crystalline Oscillatoria agardhii agglutinin (OAA), we demonstrate that judiciously designed 2D and 3D ¹⁹F-based dipolar correlation experiments such as (H)CF, (H)CHF, and FF can yield interatomic distances in the 8–16 Å range. Incorporation of fluorine-based restraints into structure calculation improved the precision of Trp side chain conformations as well as regions in the protein around the fluorine containing residues, with notable improvements observed for residues in proximity to the Trp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops, which lacked sufficient long-range ¹³C-¹³C distance restraints. Our work highlights the use of fluorine and ¹⁹F fast MAS NMR spectroscopy as a powerful structural biology tool.