FUNCTIONAL LABELING OF CYTOCHROME C BY H EXCHANGE & NMR

通常认为，式（I）的酰胺NH的H-交换行为是不稳定的。 蛋白质可以产生关于结构，结构变化， 整个蛋白质的结构动力学，但这种能力并没有 但已经实现了。 我们已经开发了差分H-交换方法， 可以标记任何蛋白质的那些部分， 相互作用（变构变化、小分子或大分子结合等）。 核磁共振的最新进展使人们有可能能够 识别蛋白质如细胞色素c中的许多酰胺NH共振， 并测量它们各自的汇率。 HX和 核磁共振技术将为蛋白质化学研究提供最有力的工具 问题研究 我们提议开始发展这种能力。 选择性 将使用标记方法，用完全ND交换的蛋白质样品， 把H放在细胞色素c的那些片段上， 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）共振使用2D NMR技术补充我们的 选择性氚标记和肽HPLC分离方法。 我们进一步怀疑，测量H-汇率的限制， 像这样的NH集将区分不同的模型， 内部动力学运动建议确定蛋白质H-交换 流程. 如果局部展开模型得到证实， (at至少对于a-螺旋），那么像这样的实验也将能够 量化单个片段对蛋白质功能的贡献， 自由能源的概念。 我们希望通过比较氧化还原， 具有不同氧化还原的相关细胞色素中的敏感H交换变化 潜力

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.