SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

这个项目的总体目标是确定 掩埋在折叠蛋白质基质中的原子被 溶剂可及的。这是通过氢同位素交换来测量的。 溶剂型氢原子与不稳定蛋白质质子的动力学 它们是肽酰胺质子。氢同位素交换动力学研究 折叠蛋白质中的多肽NN分布在6-10个数量级 震级。交换最快的NH是在表面上，而 NH交换最慢的往往是在Beta-Sheet的埋藏部分。 尽管埋藏的NH的交换比模型化合物慢，但它们的有限 交换率表明，天然蛋白质中的内部原子有一些 暴露在溶剂中的可能性。这立即意味着 蛋白质波动，使紧密堆积、掩埋的原子变得容易接近 溶剂型。 最近，我们实验室有了一个令人兴奋的发现，即交易所 牛胰酶抑制剂(BPTI)表面氨基转移速率的研究 比模型化合物慢3倍到2000倍，即它们的pH值 值的变化超过2个pH单位，有些值的PHmin小于 通常认为表面的NH原子(不是氢键) 并具有有限的静态可访问性)汇率可与 模型多肽中的那些。汇率和pH值与 然后，模型化合物就表现出了特殊的兴趣，反映了 蛋白质-溶剂界面的动态结构。 这项研究提案的具体目的是：1)描述 具有中间交换的BPTI中NH‘s的氢交换动力学 率，2)测定胰酶原和胰酶原/Ile-Val的作用 对BPTI NH汇率的约束3)比较汇率 各BPTI NH解决方案中的汇率 晶体和粉末形式，以及4)测量交换动力学 用溶剂水掩埋在BPTI-胰酶复合体中的水。

The general goal of this project is to determine the internal motions of proteins by which atoms buried in the matrix of folded proteins are accessible to solvent. This is measured by the hydrogen isotope exchange kinetics of solvent hydrogen atoms with labile protein protons, most of which are peptide amide protons. The hydrogen isotope exchange kinetics of peptide NN's in folded proteins are distributed over 6-10 orders of magnitude. The most rapidly exchangeing NH's are on the surface, and the slowest exchanging NH's tend to be in buried sections of Beta -sheet. Although buried NH's exchange slower than model compounds, their finite exchange rates demonstrate that interior atoms in native proteins have some probability of being exposed to solvent. This immediately implies that the protein fluctuates to render tightly packed, buried atoms accessible to solvent. Recently our laboratory has made the exciting findings that the exchange rates of NH's on the surface of bovine pancreatic trypsin inhibitor (BPTI) vary from 3-fold to 2000-fold slower than model compounds, that their pHmin values vary over greater than 2 pH units, and that some have pHmin of less than 1. It has been commonly assumed that surface NH atoms (not H-bonded and with finite static accessibility) exchange with rates comparable to those in model peptides. The deviation in exchange rates and pHmin from model compounds then taken on special interest as a reflection of the dynamic structure of the protein-solvent interface. The specific aims of this research proposal are 1) to characterize the hydrogen exchange kinetics of NH's in BPTI with intermediate exchange rates, 2) to measure the effect of trypsinogen and trypsinogen/Ile-Val binding on the exchange rates of BPTI NH's 3) to compare the exchange rate of individual BPTI NH's in solution with their exchange rates in the crystalline and powder forms, and 4) to measure the exchange kinetics of water buried in the BPTI-trypsin complex with solvent water.