Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
J Am Chem Soc. 2009 Dec 30;131(51):18556-62. doi: 10.1021/ja907911y.
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.
我们描述了一种新的 NMR 方法，用于监测蛋白质中天冬氨酸残基的羟基的个别氢交换速率。该方法利用 (2S,3R)-[β(2),ε(1,2)-(2)H3;0,α,β,zeta-(13)C4;(15)N]-Tyr, zeta-SAIL Tyr，通过 7-8 Hz 的 (1)H(delta)-(13)C(zeta)自旋偶合间接 (1)H 检测，或通过直接 (13)C(zeta)观察，有效地检测和分配 Tyr 环的 (13)C(zeta)信号。比较溶解在 H2O 和 D2O 中的 18.2 kDa 蛋白 EPPIb 的三个 Tyr 残基的 (13)C(zeta)化学位移，发现 D2O 中的三个 (13)C(zeta)信号均比 H2O 中的信号高约 0.13 ppm（在 150.9 MHz 下约为 20 Hz）。然而，在 H2O/D2O（1:1）混合物中，三个 (13)C(zeta)信号之一在平均化学位移处显示为单个峰，而另外两个在 H2O 和 D2O 中的化学位移完全相同，在 40 摄氏度、50 mM 磷酸盐缓冲液（pH 6.6）中。这三个峰分别被分配给 Tyr-36、Tyr-120 和 Tyr-30，从低到高的化学位移。结果表明，Tyr-120 的羟基质子交换速度快于几毫秒，而 Tyr-30 和 Tyr-36 的交换速度较慢。在这些条件下，Tyr-30 羟基质子的交换速率 k(ex) 通过 (13)C NMR 交换光谱 (EXSY)确定为 9.2 +/- 1.1 s(-1)。然而，Tyr-36 羟基质子的交换速度太慢，无法通过 EXSY 确定。这些羟基质子交换速率之间的显著差异与它们在蛋白质中的相对溶剂可及性和与 Tyr 羟基相关的氢键密切相关。
