Measurement of histidine pKa values and tautomer populations in invisible protein states
Measurement of histidine pKa values and tautomer populations in invisible protein states
复制标题
DOI：
10.1073/pnas.1400577111
复制
发表时间：
2014-04-29
影响因子：
11.1
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Hansen, Alexandar L.;Kay, Lewis E.
组氨酸咪唑侧链在蛋白质功能和稳定性中起着关键作用。它的催化作用的重要性是强调了这样一个事实，即组氨酸是本地化的活性位点在所有酶的50%左右。核磁共振波谱已成为组氨酸侧链研究的重要工具，通过测量位点特异性pK（a）和互变异构体群体。迄今为止，这样的研究已被限制在可观察到的蛋白质基态，然而，组氨酸静电在蛋白质的功能和稳定性的作用的一个完整的理解，需要类似的调查扩展到罕见的，短暂形成的构象填充的能量景观，但往往是“看不见的”在标准的NMR光谱。在这里，我们提出了NMR实验和一个简单的策略，研究这种构象激发态的基础上测量组氨酸C-13（γ），C-13（δ 2）的化学位移和H-1（γ）-C-13（δ）的单键标量耦合。首先验证该方法，然后用于获得大肠杆菌素E7免疫蛋白的不可见的途径上折叠中间体的组氨酸残基的pK（a）值和互变异构体分布。我们的研究结果表明，侧链的H40和H47暴露在中间状态，并进行重大的构象重排折叠过程中的天然结构。此外，pK（a）值解释了在pH 5.5-6.5范围内天然状态和中间状态之间的pH依赖性稳定性差异，并且它们表明咪唑去质子化不是该蛋白质折叠的障碍。
The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in similar to 50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK(a)s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine C-13(gamma), C-13(delta 2) chemical shifts and H-1(epsilon)-C-13(epsilon) one-bond scalar couplings. The methodology is first validated and then used to obtain pK(a) values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pK(a) values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.