Hydrogen exchange rate of tyrosine hydroxyl groups in proteins as studied by the deuterium isotope effect on Cζ chemical shifts

We describe a new NMR method for monitoring the individual hydrogen exchange rates of the hydroxyl groups of tyrosine (Tyr) residues in proteins. The method utilizes (2S,3R)-[β₂,ε_1,2- ²H₃;0,α,β,ζ-¹³C ₄;¹⁵N]-Tyr, ζ-SAIL Tyr, to detect and assign the ¹³C_ζ signals of Tyr rings efficiently, either by indirect ¹H-detection through 7-8 Hz ¹H _δ-¹³C_ζ spin couplings or by direct ¹³C_ζ observation. A comparison of the ¹³C_ζ chemical shifts of three Tyr residues of an 18.2 kDa protein, EPPIb, dissolved in H₂O and D₂O, revealed that all three ¹³C_ζ signals in D₂O appeared at ∼0.13 ppm (∼20 Hz at 150.9 MHz) higher than those in H ₂O. In a H₂O/D₂O (1:1) mixture, however, one of the three signals for ¹³C_ζ appeared as a single peak at the averaged chemical shifts, and the other two appeared as double peaks at exactly the same chemical shifts in H₂O and D₂O, in 50 mM phosphate buffer (pH 6.6) at 40 °C. These three peaks were assigned to Tyr-36, Tyr-120, and Tyr-30, from the lower to higher chemical shifts, respectively. The results indicate that the hydroxyl proton of Tyr-120 exchanges faster than a few milliseconds, whereas those of Tyr-30 and Tyr-36 exchange more slowly. The exchange rate of the Tyr-30 hydroxyl proton, k_ex, under these conditions was determined by ¹³C NMR exchange spectroscopy (EXSY) to be 9.2 ± 1.1 s^-1. The Tyr-36 hydroxyl proton, however, exchanges too slowly to be determined by EXSY. These profound differences among the hydroxyl proton exchange rates are closely related to their relative solvent accessibility and the hydrogen bonds associated with the Tyr hydroxyl groups in proteins.