Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, Idaho 83725-1520, United States.
J Phys Chem B. 2013 Mar 7;117(9):2653-61. doi: 10.1021/jp3117227. Epub 2013 Feb 25.
α-Conotoxin MII (α-CTxMII) is a potent and selective peptide antagonist of neuronal nicotinic acetylcholine receptors (nAChR's). Studies have shown that His9 and His12 are significant determinants of toxin binding affinity for nAChR, while Glu11 may dictate differential toxin affinity between nAChR isoforms. The protonation state of these histidine residues and therefore the charge on the α-CTx may contribute to the observed differences in binding affinity and selectivity. In this study, we assess the pH dependence of the protonation state of His9 and His12 by (1)H NMR spectroscopy and constant pH molecular dynamics (CpHMD) in α-CTxMII, α-CTxMII[E11A], and the triple mutant, α-CTxMII[N5R:E11A:H12K]. The E11A mutation does not significantly perturb the pKa of His9 or His12, while N5R:E11A:H12K results in a significant decrease in the pKa value of His9. The pKa values predicted by CpHMD simulations are in good agreement with (1)H NMR spectroscopy, with a mean absolute deviation from experiment of 0.3 pKa units. These results support the use of CpHMD as an efficient and inexpensive predictive tool to determine pKa values and structural features of small peptides critical to their function.
α-芋螺毒素 MII（α-CTxMII）是一种作用于烟碱型乙酰胆碱受体（nAChR）的高效且选择性的多肽拮抗剂。研究表明，组氨酸残基 His9 和 His12 是决定毒素与 nAChR 亲和力的关键因素，而 Glu11 可能决定了毒素与不同 nAChR 亚型之间的亲和力差异。这些组氨酸残基的质子化状态，以及 α-CTx 的电荷，可能有助于解释观察到的结合亲和力和选择性的差异。在这项研究中，我们通过（1）H NMR 光谱和 α-CTxMII、α-CTxMII[E11A]和三重突变体 α-CTxMII[N5R:E11A:H12K]中的恒 pH 分子动力学（CpHMD）评估了 His9 和 His12 的质子化状态对 pH 的依赖性。E11A 突变对 His9 或 His12 的 pKa 值没有显著影响，而 N5R:E11A:H12K 导致 His9 的 pKa 值显著降低。CpHMD 模拟预测的 pKa 值与（1）H NMR 光谱非常吻合，与实验值的平均绝对偏差为 0.3 pKa 单位。这些结果支持使用 CpHMD 作为一种高效且廉价的预测工具来确定对其功能至关重要的小肽的 pKa 值和结构特征。
