FUNCTIONAL LABELING OF CYTOCHROME C BY H EXCHANGE & NMR

这项工作将使用氢交换(HX)、核磁共振 (核磁共振)、定点突变、理论计算和其他 研究蛋白质结构问题的物理和化学方法， 动力和功能。已经开始的项目针对的是 HX行为的结构基础、蛋白质动态折叠、平衡 折叠中间体和蛋白质静电学。 支撑蛋白质HX行为的结构机制将是 学习。正在使用现场定向技术来研究局部尺度效应 重组大鼠细胞色素C的突变。这些结果也将 定量评价单个氨基酸的贡献 相互作用对蛋白质结构稳定的影响。研究更大范围的问题 ，我们收集了细胞色素C在不同功能和 四种不同化学修饰形式对细胞色素C的络合作用 蛋白质。这项工作中记录的详细的氢交换模式和 在现有的关于蛋白质和多肽系统的出版工作中， 分析过了。在这些分析中，结构对氢交换的贡献 将更清楚地显示在我们清除残留物的新能力上- 特定的化学变量。 计划进行蛋白质折叠实验，以确定 抑制Cyt c折叠，导致折叠异质性。这些残留物- 依赖的屏障将通过诱变和/或化学物质来消除 操纵已经奏效了。快速折叠将在 由此产生的构造。然后我们将尝试填充和研究折叠 通过诱变而在动力学上不可见的中间体 重新设置新的障碍。为了研究折叠中的限速步骤， 我们将以细胞色素T的平衡折叠中间体为起始原料 C，我们之前已经将其描述为处于不同的阶段 正在展开。在这项工作中，将使用特殊的方法来区分 本机与前本机与前本机的时间分辨外观 表格。 核磁共振检测的HX方法将用于获得站点解析的地图 高电荷细胞表面周围的静电势 C分子。场强分布将由场地进一步操控- 定向残基变化。这些结果将与 现有的蛋白质静电学理论模型可以提高蛋白质的静电性 模型，并帮助理解静电在 修饰蛋白质HX的行为。 将进行工作，以确定部分结构化的现实 我们在前文中提出的细胞色素C的平衡折叠中间体 工作。

This work will use hydrogen exchange (HX), nuclear magnetic resonance (NMR), site-directed mutagenesis, theoretical calculations, and other physical and chemical approaches to study problems in protein structure, dynamics, and function. Projects already begun are directed at the structural bases of HX behavior, kinetic protein folding, equilibrium folding intermediates and protein electrostatics. The structural mechanisms that underlie protein HX behavior will be studied. Local scale effects are being studied using site-directed mutations in recombinant rat cyt c. These results will also quantitatively evaluate the contribution of individual amino acid interactions to protein structural stabilization. To study larger scale effects, we have gathered HX data on cyt c in different functional and chemically modified forms and on cyt c complexed with four different proteins. The detailed H- exchange patterns recorded in this work and in available published work on protein and polypeptide systems will be analyzed. In these analyses the structural contribution to H-exchange will be more clearly displayed by our new ability to remove residue- specific chemical variables. Protein folding experiments are planned to identify the barriers that inhibit cyt c folding and lead to folding heterogeneity. These residue- dependent barriers will be removed by mutagenesis and/or by chemical manipulations already worked out. Fast folding will be studied in the resulting constructs. We will then attempt to populate and study folding intermediates that are otherwise kinetically invisible by mutagenically reinserting new barriers. To study the rate-limiting step in folding, we will use as starting material equilibrium folding intermediates of cyt c that we have characterized previously to be in different stages of unfolding. In this work special approaches will be used to distinguish the time-resolved appearance of native vs. pre-native vs. pre-native forms. An NMR-detected HX method will be used to obtain a site-resolved map of the electrostatic potential around the surface of the highly charged cyt c molecule. The field distribution will be further manipulated by site- directed residue changes. These results will be used in concert with available theoretical models of protein electrostatics to improve the models and also to help understand the role that electrostatics plays in modifying protein HX behavior. Work will be done to determine the reality of a partially structured equilibrium folding intermediate of cyt c that we proposed in previous work.