SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

蛋白质中的单质子交换动力学

• 批准号: 3273758
• 负责人: CLARE K WOODWARD
• 金额: $ 17.77万
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3273758
• 关键词: acidity /alkalinity; calorimetry; chemical reaction; circular dichroism; crosslink; crystallization; deuterium; hydrogen transport; isomorphous substitution; molecular dynamics; molecular rearrangement; nuclear magnetic resonance spectroscopy; protein denaturation; protein folding; protein structure; proteolysis; site directed mutagenesis; thermodynamics; trypsin inhibitors

Our specific aims are twofold, to continue our studies on the mechanism of protein hydrogen exchange, and to determine the relationship between internal mobility and thermodynamic stability of various submolecular domains of BPTI. These are highly interrelated; conclusions from each area of study have a direct bearing on the interpretation of results from the other. Hydrogen exchange in proteins refers to the isotype exchange kinetics of a protein NH hydrogen with solvent hydrogen. The exchange rate is a function of internal protein motions that expose the exchanging NH to solvent. Historically, hydrogen exchange provided the first experimental insight into the dynamical aspect of protein structure. We measure exchange rates of assigned NH protons by 1H NMR. OUr lab has produced a number of significant contributions to the basic understanding of the mechanism of hydrogen exchange in proteins. We propose to continue this avenue of inquiry, using the model protein bovine pancreatic trypsin inhibitor (BPTI). In addition, we are probing the relationship between protein dynamic structure and protein thermodynamic stability. 1) The mechanism of hydrogen exchange in proteins involves two central question. What structural factors account for slowing of exchange rates in proteins compared to equivalent extended peptides? What kinds of conformational fluctuations lead to the exposure of buried NH groups to solvent when the protein is in the folded state? We study these questions by determining the basic kinetics of the exchange reaction, namely, the acid and base exchange rate constants, and their variation with temperature, pH and ionic strength for BPTI in solution and in crystals. We use BPTI variants obtained by site directed mutagenesis to probe the basic chemical and conformational mechanism of protein hydrogen exchange. 2) What is the relationship between protein internal mobility and thermodynamic stability? Submolecular packing domains, or microdomains, have been defined based on hydrogen exchange properties. These are the "knot", "matrix" and "surface". The knot is the rigid hydrophobic core of atoms that pack the very slowest exchanging protons. The matrix is composed of the more flexible regions of the buried interior containing more rapidly exchanging buried NH's. The matrix in BPTI, we find, had two components, a) the matrix loops comprised of long, overlapping aperiodic segments enclosing buried water molecules, and b) a hinge region between the core and the matrix. The surface contains the NH's that are exposed to solvent and not intramolecularly H-bonded. We use site-directed mutagenesis to selectively perturb the core, matrix, or surface of BPTI. Then we measure changes in internal flexibility by hydrogen exchange and proteolytic susceptibility. The thermodynamics of the cooperative denaturation are determined by calorimetry and circular dichroism. Time average structures of mutant BPTI's are modeled using NMr distances and constrained molecular dynamics. With these approaches we ask a number of questions regarding the relationship between protein dynamics and thermodynamics.

我们的具体目标是双重的，继续我们的研究机制， 蛋白质氢交换，并确定 各种亚分子内部流动性和热力学稳定性 BPTI的域。 这些是高度相关的;每个领域的结论 研究结果的解释有直接的关系， 其他. 蛋白质中的氢交换是指同种型交换 蛋白质NH氢与溶剂氢的动力学。 汇率 是内部蛋白质运动的函数， 溶剂后 历史上，氢交换提供了第一个实验性的 深入了解蛋白质结构的动力学方面。 我们测量 通过1H NMR测定指定NH质子的交换速率。 我们的实验室制作了一个 一些重大贡献的基本认识， 蛋白质中的氢交换机制。 我们建议继续这样做 采用牛胰蛋白酶模型蛋白， 抑制剂（BPTI）。 此外，我们还在探索 蛋白质动力学结构和蛋白质热力学稳定性。 1)蛋白质中氢交换的机制涉及两个中心 问题 哪些结构性因素导致了2008年汇率放缓 蛋白质与等效的延伸肽相比？ 什么样的 构象波动导致埋藏的NH基团暴露于 当蛋白质处于折叠状态时，溶剂是什么？ 我们研究这些问题 通过确定交换反应的基本动力学，即， 酸和碱交换速率常数，以及它们随 温度、pH和离子强度。 我们使用通过定点诱变获得的BPTI变体来探测BPTI的突变。 蛋白质氢交换的基本化学和构象机制。 2)蛋白质内部流动性与 热力学稳定性？ 亚分子堆积域，或微域， 已经基于氢交换性质定义。 这些都是 “结”、“基质”和“表面”。 结是刚性疏水核心， 质子交换最慢的原子。 所述基质是 由埋在地下的内部更柔软的区域组成， 更快地交换被掩埋的NH。 我们发现，BPTI中的矩阵有两个 成分，a）由长的、重叠的非周期性的 封闭埋藏水分子的片段，和B）铰链区， 核心和母体 表面含有氨， 溶剂，而不是分子内氢键。 我们使用网站导向 诱变以选择性地扰动BPTI的核心、基质或表面。 然后，我们通过氢交换测量内部柔性的变化， 蛋白水解敏感性 合作社的热力学 通过量热法和圆二色性测定变性。 时间 使用NMR距离对突变BPTI的平均结构进行建模， 约束分子动力学 通过这些方法，我们询问了一些 关于蛋白质动力学和 热力学