Visualizing and Braking Protein Ring Flips with Difluorotyrosines

Aromatic ring-flip dynamics are hallmarks of concerted protein "breathing" motions that are essential for biological function. However, accurately measuring-and especially modulating-ring-flip dynamics remains challenging, particularly in living cells. Here, we demonstrate that ¹⁹F NMR is powerful for quantifying ring flips on the μs-s time scale. Using cost-effective difluorotyrosine probes, 3,5-F₂Y and 2,6-F₂Y, we detect the previously hidden tyrosine ring flips (τ_flip ≈ 15 μs) in ubiquitin. Importantly, 3,5-F₂Y is a largely nonperturbing ring-flip probe while 2,6-F₂Y acts as an effective "brake", slowing ring flips by ∼10³-fold in GB1 and HPr. We further evaluate how macromolecular crowding and the intracellular environment modulate ring-flip dynamics. Our work opens new avenues for measuring and modulating ring flips in vitro and in living cells.