Characterizing Tyrosine Ring Flips in Proteins by ¹⁹ F NMR

Aromatic ring flip dynamics are essential for elucidating the catalytic mechanism of enzyme, protein binding, and the “breathing” motions of proteins. NMR is the primary method for measuring aromatic ring flip rates in proteins at the residue level, but it typically requires extreme conditions such as high pressure or low temperature to slow down the ring flips to a detectable rate on the NMR time scale. Consequently, ring flip rates have only been measured for a limited number of proteins due to the lack of simple method under mild conditions. Here we report a novel ¹⁹ F NMR method utilizing 2,6-difluoro-tyrosine (2,6-F ₂ Y) substitution to measure the ring flip rate of tyrosine residues in vitro and in living cells. We validated the effectiveness of method on three proteins, the B1 domain of protein G (GB1), ubiquitin (UBQ) and histidine-containing phosphocarrier protein (HPr). The results demonstrated that this ¹⁹ F NMR approach can quantify the ring flip on timescales from microseconds to seconds under mild experimental conditions. For the first time, we measured the tyrosine ring flip dynamics of HPr in three types of living cells: E. coli , A2780 cells and X. laevis oocytes. Our findings reveal that tyrosine ring flip dynamics are remarkably affected by cellular environment and are cell-type dependent. The strong interactions and the low activation enthalpy are associated with fast ring flipping. Our study provides a simple, convenient and efficient method for characterizing tyrosine ring flip in proteins both in vitro and in cells, and it also opens a new avenue for measuring the ring flip rates in proteins in complex biological environments.