SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

蛋白质中的单质子交换动力学

• 批准号: 3273759
• 负责人: CLARE K WOODWARD
• 金额: $ 14.86万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3273759
• 关键词: acidity /alkalinity; chemical reaction; conformation; crosslink; hydrogen transport; isomorphous substitution; molecular rearrangement; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; stop flow technique; thermodynamics; trypsin inhibitors

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.

该项目的一般目标是确定 原子埋在折叠蛋白基质中的蛋白质是 溶剂可访问。 这是通过氢同位素交换来衡量的 具有不稳定蛋白质质子的溶剂氢原子的动力学，大多数 是肽酰胺质子。 氢同位素交换动力学 折叠蛋白中的肽NN的分布在6-10个顺序上 震级。 最快的NH交换在地面上， 最慢的NH交换往往是在beta -sheet的掩埋部分中。 尽管埋藏NH的交换比模型化合物慢，但它们的有限 汇率表明，天然蛋白质中的内部原子有一些 暴露于溶剂的概率。 这立即意味着 蛋白质波动以使堆积紧紧，埋葬原子可及 溶剂。 最近，我们的实验室提出了令人兴奋的发现 NH在牛胰腺胰蛋白酶抑制剂（BPTI）表面的速率 比模型化合物慢3倍至2000倍不等 值在大于2个pH单元的情况下变化，有些具有较小的Phmin 超过1。通常假定表面NH原子（未h键 并具有有限的静态可访问性）汇率，费率可与 在模型肽中的那些。 汇率和phmin的偏差 然后，模型化合物特别感兴趣作为反映 蛋白质溶剂界面的动态结构。 该研究建议的具体目的是1）表征 NH的氢交换动力学在BPTI中与中间交换 速率，2）测量胰蛋白酶原和胰蛋白酶原/ILE-VAL的作用 对BPTI NH的汇率3）进行约束，以比较汇率 个人bpti nh的解决方案的汇率 结晶和粉末形式，以及4）测量交换动力学 用溶剂水将水埋在Bpti-trypsin综合体中。