FUNCTIONAL LABELING OF CYTOCHROME C BY H EXCHANGE & NMR

通过 H 交换对细胞色素 C 进行功能标记

• 批准号: 2176342
• 负责人: S. Walter ENGLANDER
• 金额: $ 28.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176342
• 关键词: analytical chemistry; chemical kinetics; cytochrome c; hydrogen; intermolecular interaction; ionic bond; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein folding; protein structure function; site directed mutagenesis

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.

这项工作将使用氢交换（HX）、核磁共振 (NMR)、定点突变、理论计算等 研究蛋白质结构问题的物理和化学方法， 动力学和功能。 已经开始的项目针对的是 HX 行为的结构基础、蛋白质折叠动力学、平衡 折叠中间体和蛋白质静电学。 蛋白质 HX 行为的结构机制是 研究过。 正在使用定点研究局部规模效应 重组大鼠细胞色素突变 c. 这些结果也将 定量评估单个氨基酸的贡献 与蛋白质结构稳定性的相互作用。 研究更大规模 影响，我们收集了不同功能和细胞色素 c 的 HX 数据 化学修饰形式和细胞色素 c 与四种不同的复合物 蛋白质。 本工作中记录的详细 H-交换模式和 在有关蛋白质和多肽系统的现有已发表的著作中将是 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展示- 具体的化学变量。 计划进行蛋白质折叠实验来确定蛋白质折叠的障碍 抑制细胞色素c折叠并导致折叠异质性。 这些残留物—— 依赖性障碍将通过诱变和/或化学方法消除 操纵已经奏效。 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， 我们将使用细胞色素的平衡折叠中间体作为起始材料 c 我们之前已经将其描述为处于不同阶段 展开。 在这项工作中，将使用特殊方法来区分 原生与原生前与原生前的时间分辨外观 形式。 NMR 检测的 HX 方法将用于获得位点解析图 高电荷细胞色素表面周围的静电势 c 分子。 现场分布将由现场进一步操纵 定向残留变化。 这些结果将与 可用的蛋白质静电学理论模型来改进 模型，还可以帮助理解静电在其中所扮演的角色 改变蛋白质 HX 行为。 将开展工作以确定部分结构化的现实 我们在之前提出的 cyt c 的平衡折叠中间体 工作。