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

基本信息

批准号：
3280230
负责人：
S. Walter ENGLANDER
金额：
$ 26.59万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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

项目摘要

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结合起来 NMR 测量和定点突变能力探索蛋白质结构和功能的问题。测试蛋白质是马心细胞色素c。在本次活动的第一阶段工作中，还原和氧化细胞色素 c 中的质子共振是分配并制定了衡量交换的方法所有酰胺 NH 质子和一些侧链质子。这种能力将被用来研究以下领域的基本问题：通过获得质子解析的 H 交换数据来进行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结构和动力学的变化突变将通过 H 交换和 2D NMR 研究光谱学。快速交换质子的测量，不涉及结构，将有助于校准静电效应并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局部的剩余结构问题正在展开蛋白质形式。广泛的中间范围的氢交换将进行检查以了解局部协作动态运动，稳定相互作用和蛋白质设计。实验于细胞色素 c 不同功能形式的进展将持续。还原态和氧化态之间的 H 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。 H交换细胞色素 c 的测量将在其与细胞色素c过氧化物酶。不断增长的位点解析 HX 数据库，包含 100 多个探针贯穿细胞色素 c 结构的各种结构点和功能状态，预计将揭示种类 HX 流程背后的结构性波动，有助于开发此类信息对研究的效用结构、动力学、能量以及功能相关的变化在这些参数中。