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: 3280233
• 负责人: S. Walter ENGLANDER
• 金额: $ 8.23万
• 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 1988-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280233
• 关键词: allosteric site; amides; chemical structure function; chemical synthesis; cytochrome c; electrical potential; high performance liquid chromatography; hydrogen; hydrogen bond; mitochondria; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; radiotracer; thermodynamics; allosteric site; amides; chemical structure function; chemical synthesis; cytochrome c; electrical potential; high performance liquid chromatography; hydrogen; hydrogen bond; mitochondria; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; radiotracer; thermodynamics

It is generally recognized that the H-exchange behavior of the amide NH of a protein can yield information about structure, structure change, and structural dynamics all through the protein, but this capability has not yet been achieved. We have developed differential H-exchange methods that can label just those parts of any protein that participate in any interaction (allosteric change, small or large molecule binding, etc.). Recent advances in NMR now make it probable that one will be able to recognize many of the amide NH resonances in a protein like cytochrome c, and measure their individual exchange rates. The coupling of the HX and NMR capabilities would provide a most powerful tool for protein chemical studies. We propose to begin to develop this capability. The selective labelling methods will be used, with samples of fully ND-exchanged protein, to put H on just those segments of cytochrome c that act to adjust the redox potential of the heme, or that interact with the oxidase, or the reductase, etc. Then we will attempt to identify the sensitive (protonated) resonances using 2D NMR techniques supplemented by our selective tritium-labelling and peptide HPLC separation methods. We further suspect that measurement of the H-exchange rates of restricted sets of NH like these will distinguish between alternative models for the internal dynamical motions suggested to determine protein H-exchange processes. If the local unfolding model is confirmed, as now seems likely (at least for a-helices), then experiments like these will also be able to quantify the contribution of individual segments to protein function in terms of free energy. We hope to check this capability by comparing redox sensitive H-exchange changes in related cytochromes having differing redox potentials.

人们普遍认为，酰胺NH的H-交换行为 蛋白质可以产生有关结构，结构变化和的信息 整个蛋白质的结构动力学，但这种能力尚未 尚未实现。 我们已经开发了不同的H-交换方法 只能标记任何参与任何蛋白质的部分 相互作用（变构变化，小或大分子结合等）。 现在，NMR的最新进展使得很可能 识别许多酰胺NH在细胞色素C等蛋白质中的共振， 并衡量他们的个人汇率。 HX的耦合和 NMR功能将为蛋白质化学提供最强大的工具 研究。 我们建议开始发展这一能力。 选择性 将使用标记方法，并使用完全ND交换蛋白的样品， 仅将H放在细胞色素c的那些片段上，以调整 血红素的氧化还原电位，或与氧化酶相互作用或 还原酶等。然后，我们将尝试识别敏感的 （质子化的）使用2D NMR技术补充了我们的共振 选择性tri列标签和肽HPLC分离方法。 我们进一步怀疑限制的H交换率的测量 这样的NH集将区分 建议确定蛋白质H-交换的内部动力学运动 过程。 如果确认本地展开模型，那么现在似乎很可能 （至少对于A螺旋），然后类似的实验也将能够 量化单个片段对蛋白质功能的贡献 自由能的条款。 我们希望通过比较氧化还原来检查此功能 具有不同氧化还原的相关细胞色素的敏感H-交换变化 潜力。