Proton magnetic resonance study of Streptomyces subtilisin inhibitor. pH titration and assignments of individual tyrosyl resonances.
Proton magnetic resonance study of Streptomyces subtilisin inhibitor. pH titration and assignments of individual tyrosyl resonances.
复制标题
链霉菌枯草杆菌蛋白酶抑制剂的质子磁共振研究。
作者：
S. Fujii;K. Akasaka;H. Hatano
本文报道了 25 摄氏度下的 pH 滴定，以及通过高分辨率 1 H NMR 光谱对链霉菌枯草杆菌蛋白酶抑制剂 (Mr 23000) 每个相同亚基的三个酪氨酰残基进行芳香质子共振的归属。特定酪氨酰共振的完整归属是基于用四硝基甲烷对酪氨酰残基进行差异化学修饰的结果，然后进行肽分析，与晶体结构的知识无关。在25℃下，在2H2O溶液中，Tyr-7、-75和-93的pKa值分别测定为10.95、11.8和大于或等于12.6，而硝化Tyr-7和-75的pKa值分别测定为7.3和7.9。 Tyr-93 在正常条件下不可硝化。 Tyr-93 的强硝化抗性，以及极高的 pKa 值和中性 pH 下环质子共振的高场位移，强烈表明 Tyr-93 作为质子供体参与氢键结合。 Tyr-7 比 Tyr-75 更容易用四硝基甲烷修饰，尽管在晶体中 Tyr-75 比 Tyr-7 暴露得更多。结果加上 Tyr-75 的 pKa 值显着高于正常酪氨酸，表明 Tyr-75 在溶液中的微环境比在固体中受到更多限制。这些结果表明，溶液中蛋白质的结构细节可能与蛋白质刚性疏水区域中晶体中的结构细节非常相似，但蛋白质表面区域的局部结构在溶液和晶体之间可能有很大不同。
This paper reports the pH titration at 25 degrees C and the assignment of aromatic proton resonances of three tyrosyl residues of each identical subunit of Streptomyces subtilisin inhibitor (Mr 23000) by high-resolution 1H NMR spectroscopy. The complete assignments of the specific tyrosyl resonances were made based on the results of the differential chemical modification of the tyrosyl residues with tetranitromethane followed by peptide analysis, independently of the knowledge of the crystal structure. pKa values of Tyr-7, -75, and -93 were determined in a 2H2O solution to be 10.95, 11.8, and greater than or equal to 12.6, respectively, at 25 degrees C, whereas pKa values of nitrated Tyr-7 and -75 were determined to be 7.3 and 7.9, respectively. Tyr-93 was not nitratable under normal conditions. The strong resistance to nitration, together with the extremely high pKa value and the high-field shifted positions of the ring proton resonances of Tyr-93 at neutral pH, strongly suggests that Tyr-93 takes part in a hydrogen bonding as a proton donor. Tyr-7 is more easily modified with tetranitromethane than Tyr-75, although in the crystal Tyr-75 is more exposed than Tyr-7. The result, together with the pKa value of Tyr-75 significantly higher than that of a normal tyrosine, indicates that the microenvironment of Tyr-75 is more restricted in solution than in solid. These results imply that structural details of a protein may be quite similar in solution to those in the crystal in the rigid hydrophobic region of the protein but that in the surface region of the protein local structures may well differ between the solution and the crystal.