Solid-State NMR ¹⁹F-¹H-¹⁵N Correlation Experiments for Resonance Assignment and Distance Measurements of...

Several solid-state NMR techniques have been introduced recently to measure nanometer distances involving ¹⁹F, whose high gyromagnetic ratio makes it a potent nuclear spin for structural investigation. These solid-state NMR techniques either use ¹⁹F correlation with ¹H or ¹³C to obtain qualitative interatomic contacts or use the rotational-echo double-resonance (REDOR) pulse sequence to measure quantitative distances. However, no NMR technique is yet available for disambiguating ¹H-¹⁹F distances in multiply fluorinated proteins and protein-ligand complexes. Here, we introduce a three-dimensional (3D) ¹⁹F-¹⁵N-¹H correlation experiment that resolves the distances of multiple fluorines to their adjacent amide protons. We show that optimal polarization transfer between ¹H and ¹⁹F spins is achieved using an out-and-back ¹H-¹⁹F REDOR sequence. We demonstrate this 3D correlation experiment on the model protein GB1 and apply it to the multidrug-resistance transporter, EmrE, complexed to a tetrafluorinated substrate. This technique should be useful for resolving and assigning distance constraints in multiply fluorinated proteins, leading to significant savings of time and precious samples compared to producing several singly fluorinated samples. Moreover, the method enables structural determination of protein-ligand complexes for ligands that contain multiple fluorines.