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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)-[beta(2),epsilon(1,2)-(2)H(3);0,alpha,beta,zeta-(13)C(4);(15)N]-Tyr, zeta-SAIL Tyr, to detect and assign the (13)C(zeta) signals of Tyr rings efficiently, either by indirect (1)H-detection through 7-8 Hz (1)H(delta)-(13)C(zeta) spin couplings or by direct (13)C(zeta) observation. A comparison of the (13)C(zeta) chemical shifts of three Tyr residues of an 18.2 kDa protein, EPPIb, dissolved in H(2)O and D(2)O, revealed that all three (13)C(zeta) signals in D(2)O appeared at approximately 0.13 ppm ( approximately 20 Hz at 150.9 MHz) higher than those in H(2)O. In a H(2)O/D(2)O (1:1) mixture, however, one of the three signals for (13)C(zeta) 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 H(2)O and D(2)O, in 50 mM phosphate buffer (pH 6.6) at 40 degrees 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 (13)C 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.

The unambiguous assignment of the aromatic ring resonances in proteins has been severely hampered by the inherently poor sensitivities of the currently available methodologies developed for uniformly 13C/15N-labeled proteins. Especially, the small chemical shift differences between aromatic ring carbons and protons for phenylalanine residues in proteins have prevented the selective observation and unambiguous assignment of each signal. We have solved all of the difficulties due to the tightly coupled spin systems by preparing regio-/stereoselectively 13C/2H/15N-labeled phenylalanine (Phe) and tyrosine (Tyr) to avoid the presence of directly connected 13C-1H pairs in the aromatic rings. The superiority of the new labeling schemes for the assignment of aromatic ring signals is clearly demonstrated for a 17 kDa calcium binding protein, calmodulin.

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,ϵ1,2-2H3O,α,β,ζ-13C4;15N]-Tyr, ζ-SAlL Tyr, to detect and assign the 13Cζ signals of Tyr rings efficiently, either by indirect 1H-detection through 7-8 Hz 1Hδ-13Cζ spin couplings or by direct 13Cζ observation. A comparison of the 13Cζ chemical shifts of three Tyr residues of an 18.2 kDa protein, EPPIb, dissolved in H2O and D2O, revealed that all three 13Cζ signals in D2O appeared at ∼0.13 ppm (∼20Hz at 150.9 MHz) higher than those in H2O. In a H2O/D2O (1:1) mixture, however, one of the three signals for 13Cζ 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, kex, 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. [ABSTRACT FROM AUTHOR]