FUNCTIONAL LABELING OF CYTOCHROME C BY H-EXCHANGE & NMR

这项工作将氢交换方法学与二维 核磁共振测量与定点突变能力 追求蛋白质结构和功能方面的问题。这个测试 蛋白质是马心细胞色素c。在此的第一阶段 工作中，还原和氧化的细胞色素c中的质子共振 制定了用于测量交易所的分配和方法 在所有的酰胺、NH质子和一些侧链质子中。 这一能力将被用来研究 通过获得质子解析的氢交换数据来研究蛋白质化学 细胞色素c的各种结构形式和功能状态。 化学修饰引起的结构和动力学变化 突变将通过氢交换和2D核磁共振进行研究 光谱学。快速交换质子的测量 都不涉及结构，将有助于校准静电 影响和检验当前的静电理论。测量 最慢的氢气将用于研究全球稳定性和 全球和局部剩余结构的问题展开 蛋白质形式。交换氢气的宽广中间范围将 被检查以了解局部协同动力学运动， 稳定的相互作用和蛋白质设计。实验在 不同功能形式的细胞色素c的研究进展 继续。还原和氧化过程中氢交换的差异 形式应阐明结构之间的相互作用 能量和血红素氧化还原电势的设定。H-交换 细胞色素c的测量将在它被络合到 细胞色素c过氧化物酶。 不断增长的现场解析HX数据库，探头超过100个 点贯穿于细胞色素c结构中的各种结构 和功能状态，预计将揭示出 结构性波动是HX进程的基础，并有助于 开发这类信息在研究中的作用 结构、动力学和能量以及与功能相关的变化 在这些参数中。

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.