Hydrogen Exchange Rate of Tyrosine Hydroxyl Groups in Proteins As Studied by the Deuterium Isotope Effect on C_ζ Chemical Shifts

Abstract

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.

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

“…This effect is more consistent with a hydride transfer mechanism simply because the hydroxyl proton in Tyr residues is exchangeable with the solvent. Exchange rates in Tyr residues buried in enzyme active sites are about 9 s Ϫ1 (35), that is 3 times slower than the turnover rate. Therefore, during enzymatic assays in D 2 O, Tyr residues happened to be only partially deuterated, causing a modest isotope effect.…”

Ground-State Proton Transfer Kinetics in Green Fluorescent Protein

“…Recent work in our lab and others has utilized direct carbon detection of tyrosine isotopically labeled at position ζ [adjacent to the hydroxyl group (see the asterisks in Figure 2A)] to study the thermodynamic properties and qualitative kinetics of PT. 26,28,29 After the GFP chromophore maturation process, 13 C ζ of Tyr66 is located directly adjacent to the ionizable site and provides a convenient and nonperturbative spectator with which to follow PT. We utilize this approach to look at the widely used Superfolder GFP, 30 a particularly stable and fast-folding GFP variant, and some select circular permutants (Figure 1) to analyze the interplay between structural rigidity and PT from this new nonoptical observable.…”

Quantum Calculations Of A Large Section Of The Voltage Sensing Domain Of The Kv1.2 Channel Show That Proton Transfer, Not S4 Motion, Provides The Gating Current

“…Furthermore, the structures that transfer protons often must be considered in groups of three to five residues; one member in a group we have calculated is a phenol, tyrosine. A millisecond rate of proton exchange for a tyrosine has been shown to be possible by Takeda and coworkers 28 . These structures appear in the results of the calculations for the upper (extracellular) part of the VSD.…”