Characterizing Tyrosine Ring Flips in Proteins by ¹⁹ F NMR

Aromatic ring-flip dynamics are hallmarks of concerted protein “breathing” motions that are essential for biological function. Ring flips occur on a broad range of timescales (ns–s) and have been primarily studied by NMR spectroscopy, typically requiring expensive isotope labeling of aromatic side chains, thereby limiting such studies to a few proteins. Here, we report two novel di-fluorotyrosine probes, 3,5-F ₂ Y and 2,6-F ₂ Y, that can be incorporated into proteins cost-effectively for characterizing and modulating tyrosine ring-flip dynamics. We show that ¹⁹ F rotating-frame ( R _{1 ρ} ) relaxation dispersion is powerful for quantifying ring flips on the µs–ms timescale. Importantly, the tyrosine ring-flip rates ( k _flip ) for 3,5-F ₂ Y in GB1 and HPr are comparable to those measured by ¹ H and ¹³ C NMR studies, validating 3,5-F ₂ Y as a largely non-perturbing, native-like ring-flip probe. In contrast, 2,6-F ₂ Y acts as an effective “brake” on ring flips, enabling direct visualization and quantitative characterization of previously undetected tyrosine ring flips in ubiquitin via ¹⁹ F lineshape analysis. Furthermore, we use 2,6-F ₂ Y to assess the environmental effects on ring-flip dynamics in crowding reagents and in living X. laevis oocytes. Collectively, our study opens a new avenue for measuring and modulating ring-flip dynamics in vitro and in living cells.