Platzer Gerald, Okon Mark, McIntosh Lawrence P
Department of Biochemistry and Molecular Biology, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
J Biomol NMR. 2014 Nov;60(2-3):109-29. doi: 10.1007/s10858-014-9862-y. Epub 2014 Sep 20.
The pK a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the (1)H, (13)C, and (15)N nuclei in the seven common ionizable amino acids (X = Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain (15)N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.
多肽和蛋白质中可电离残基的pKa值和电荷状态通常通过核磁共振监测的pH滴定来确定。为了帮助解释所得的滴定数据，我们在封闭的三肽乙酰基-Gly-X-Gly-酰胺的背景下，测量了七种常见可电离氨基酸（X = 天冬氨酸、谷氨酸、组氨酸、半胱氨酸、酪氨酸、赖氨酸和精氨酸）中几乎所有的（1）H、（13）C和（15）N核的pH依赖性化学位移。丙氨酸酰胺和N-乙酰丙氨酸分别用作N端和C端的模型。这项研究共同提供了一套基本完整的pH依赖性残基内和最近邻参考化学位移，以帮助指导蛋白质pKa的测量。这些数据还应有助于在用于预测无规卷曲多肽化学位移的算法中进行pH依赖性校正。同时，还测量了组氨酸、赖氨酸和精氨酸侧链（15）N核在其带正电荷和中性状态下的氘同位素位移。连同先前发表的天冬氨酸、谷氨酸、半胱氨酸和酪氨酸的结果，这些氘同位素位移可以为定义蛋白质残基的电离状态提供补充实验证据。
