FUNCTIONAL LABELING OF CYTOCHROME C BY H EXCHANGE & NMR

这项工作将使用氢交换（HX），核磁共振 (NMR)，定点诱变，理论计算，和其他 物理和化学方法来研究蛋白质结构问题， 动力学和功能。 已经开始的项目是针对 HX行为的结构基础，蛋白质动力学折叠，平衡 折叠中间体和蛋白质静电。 蛋白质HX行为的结构机制将是 研究了 当地规模的影响正在研究使用现场导向 重组大鼠细胞色素C中的突变。 这些结果也将 定量评估单个氨基酸的贡献 蛋白质结构稳定的相互作用。 为了研究更大规模的 影响，我们收集了不同功能和 化学修饰形式和与四种不同的 proteins. 详细的H-交换模式记录在这项工作中， 在关于蛋白质和多肽系统可用的已出版的著作中， 分析了 在这些分析中，H交换的结构贡献 我们去除残留物的新能力将更清楚地体现出来- 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折叠并导致折叠异质性。 这些残留物- 依赖性屏障将通过诱变和/或化学方法去除， 操纵已经解决了。 快速折叠将在 产生的结构。 然后，我们将尝试填充和研究折叠 中间体，否则通过诱变在动力学上不可见 重新设置新的障碍。 为了研究折叠的限速步骤， 我们将使用Cyt的平衡折叠中间体作为起始材料， 我们之前已经描述了处于不同阶段的 展开 在这项工作中，将使用特殊的方法来区分 原生与原生前与原生前的时间分辨外观 forms. NMR检测的HX方法将用于获得以下的位点分辨图： 高度带电的细胞色素表面周围的静电势 c分子。 场分布将由现场进一步操纵- 定向残基变化。 这些结果将与以下内容一起使用： 可用的蛋白质静电理论模型，以提高 模型，并帮助理解静电在 修饰蛋白HX行为。 将开展工作，以确定部分结构化的 平衡折叠中间体的cyt c，我们提出了在以前的 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.