Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface.
Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface.
复制标题
牛胰蛋白酶抑制剂蛋白质-溶剂界面处肽酰胺质子的氢动力学。
DOI：
10.1016/0022-2836(85)90412-7
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发表时间：
1985
影响因子：
5.6
通讯作者：
Woodward,C
中科院分区：
文献类型：
--
作者：
Tüchsen,E;Woodward,C
牛胰蛋白酶抑制剂中 25 个交换最快的肽酰胺质子的氢交换速率常数已在跨越 pHmin（最小速率 pH）的 pH 范围内测定。其中大部分位于蛋白质表面，暴露于溶剂中，并且在晶体结构中没有氢键结合。与普遍持有的假设相反，表面 NH 基团的交换动力学并不等同于延伸构型的肽中 NH 基团的动力学。所有表面 NH 基团的交换速度都比模型肽中的 NH 基团慢，速率常数分布在两个数量级以上的范围内。此外，它们的 pH 最小值差异很大。对于大多数表面 NH 基团，pHmin 低于模型化合物，并且对于某些化合物，pHmin < 1。这些结果表明，交换事件发生时表面肽基团的局部环境与延伸肽的局部环境非常不同。基于酸催化的 O-质子化机制和静电对交换动力学影响的分析进一步表明（参见随附论文），一般来说，表面 NH 基团的交换发生在晶体结构近似的蛋白质构象中。 1 H-2 H交换率常数是从300 MHz核磁共振谱测量的，其中指定的表面N 1 H共振通过使用部分氘化的蛋白质样品来解析。在 1 至 4.5 的 pH 范围内观察到的几个表面 NH 基团的化学位移具有显着的 pH 依赖性，这反映了附近羧基的滴定。
Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pHmin, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pHminvalues vary widely. For most of the surface NH groups, pHminis lower than in model compounds and, for several, pHminis < 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The1H-2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.