FUNCTIONAL LABELING OF CYTOCHROME C BY H-EXCHANGE & NMR

这项工作将氢交换方法与二维 NMR测量和定点突变能力 研究蛋白质结构和功能的问题。 测试 蛋白质是马心细胞色素C。 在第一阶段， 工作，质子共振在还原和氧化细胞色素c， 并制定了衡量交换的方法 所有的酰胺NH质子和一些侧链质子。 这种能力将用于研究基本问题， 蛋白质化学通过获得质子分辨H-交换数据 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H-交换和2D NMR进行研究 谱 快速交换质子的测量， 不涉及结构，将有助于校准静电 影响和测试当前的静电理论。 测量 最慢的氢将被用于研究全球稳定性， 在全球和地方一级， 蛋白质形式。 交换氢的宽的中间范围将 来学习局部合作动力学运动， 稳定相互作用和蛋白质设计。 实验 细胞色素c的不同功能形式的进展将是 持续期间内的 还原态和氧化态之间的氢交换差异 形式有望阐明结构之间的相互作用， 能量和血红素氧化还原电位的设置。 氢交换 细胞色素c的测量将在其与 细胞色素c过氧化物酶 不断增长的站点解析HX数据库，超过100个探针 细胞色素c结构中的各种结构点 和功能状态，有望揭示 结构波动的基础HX过程，并帮助 开发这类信息的实用性， 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.