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 19 F NMR is powerful for quantifying ring flips on the μs–s time scale. Using cost-effective difluorotyrosine probes, 3,5-F 2 Y and 2,6-F 2 Y, we detect the previously hidden tyrosine ring flips (τ flip ≈ 15 μs) in ubiquitin. Importantly, 3,5-F 2 Y is a largely nonperturbing ring-flip probe while 2,6-F 2 Y acts as an effective “brake”, slowing ring flips by ∼10 3 -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.