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: 3280238
• 负责人: S. Walter ENGLANDER
• 金额: $ 30.65万
• 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 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280238
• 关键词: allosteric site; amides; chemical structure function; chemical synthesis; cytochrome c; deuterium; electrical potential; high performance liquid chromatography; hydrogen; hydrogen bond; hydrogen transport; membrane transport proteins; mitochondria; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; protein engineering; protein structure; radiotracer; site directed mutagenesis; thermodynamics; tritium

This work joins hydrogen exchange methodology with two dimensional NMR measurement and the capability for site-directed mutagenesis to pursue problems in protein structure and function. The test protein is horse heart cytochrome c. In the first stage of this work, proton resonances in reduced and oxidized cytochrome c were assigned and methods were developed for measurement of the exchange of all the amide NH protons and some side chain protons. This capability will be used to study the fundamental problems in protein chemistry by obtaining proton-resolved H-exchange data on various structural forms and functional states of cytochrome c. Changes in structure and dynamics due to chemical modifications and mutations will be studied by H-exchange and 2D NMR spectroscopy. Measurement of the fast exchanging protons, which are not involved in structure, will help to calibrate electrostatic effects and test current electrostatic theories. Measurement of the slowest hydrogens will be used to study global stability and the issue of remaining structure in globally and locally unfolded protein forms. The broad middle range of exchanging hydrogens will be examined to learn about locally cooperative dynamical motions, stabilizing interactions, and protein design. Experiments in progress with different functional forms of cytochrome c will be continued. H-exchange differences between the reduced and oxidized forms are expected to illuminate the interaction between structural energy and setting of the heme redox potential. H-exchange measurements on cytochrome c will be done while it is complexed to cytochrome c peroxidase. The growing library of site-resolved HX data, at over 100 probe points throughout the cytochrome c structure in various structural and functional states, is expected to reveal the kinds of structural fluctuations that underly the HX process, and help develop the utility of this kind of information for study of structure, dynamics, and energy, and functionally related changes in these parameters.

这项工作将氢交换方法与二维 NMR测量和位置诱变的能力 在蛋白质结构和功能方面进行问题。 测试 蛋白质是马心细胞色素c。 在第一阶段 工作，减少和氧化的细胞色素C中的质子共振为 分配并开发了用于测量交换的方法 在所有酰胺NH质子和某些侧链质子中。 此功能将用于研究 通过获得质子分辨的H-交换数据的蛋白质化学 细胞色素c的各种结构形式和功能状态。 由于化学修饰而导致的结构和动力学变化 并将通过H-Exchange和2D NMR研究突变 光谱法。 快速交换质子的测量 不参与结构，将有助于校准静电 效果和测试当前的静电理论。 测量 最慢的氢将用于研究全球稳定性和 全球和本地展开的剩余结构的问题 蛋白质形式。 较广泛的中间交换氢将 接受检查以了解本地合作的动力学运动， 稳定相互作用和蛋白质设计。 实验 具有不同功能形式的细胞色素C的进展将是 持续。 还原和氧化之间的H交换差异 预计形式有望阐明结构之间的相互作用 血红素氧化还原电位的能量和设置。 h-exchange 将细胞色素C的测量在复合到时将进行 细胞色素C过氧化物酶。 在100多个探针中，现场分辨的HX数据的库不断增长 各种结构中的整个细胞色素C结构的点 功能状态有望揭示 基础HX过程的结构波动，并有助于 开发此类信息的实用性以进行研究 结构，动力学和能量以及功能相关的变化 在这些参数中。