FUNCTIONAL LABELING OF CYTOCHROME C BY H EXCHANGE & NMR

一般认为，氨基NH的氢交换行为 蛋白质可以产生关于结构、结构变化和 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氢交换方法， 可以只标记任何蛋白质中参与任何 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 识别像细胞色素c这样的蛋白质中的许多酰胺NH共振， 并衡量他们各自的汇率。HX和HX的耦合 核磁共振能力将为蛋白质化学提供一个最强大的工具 学习。我们建议开始发展这一能力。精挑细选 将使用标记方法，使用完全ND交换的蛋白质样本， 将H放在细胞色素c的那些片段上，这些片段起到调节细胞色素C的作用 血红素的氧化还原电位，或与氧化酶相互作用的电位，或 还原酶等。然后我们将尝试识别敏感的 使用2D核磁共振技术的(质子化)共振，并补充了我们的 选择性氚标记和多肽高效液相分离方法。 我们进一步怀疑，对受限制的H-汇率的测量 像这样的NH集将区分不同的替代模型 内部动力学运动对蛋白质氢交换的影响 流程。如果当地的展开模型得到确认，就像现在看起来可能的那样 (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 自由能的条件。我们希望通过比较氧化还原来检查这种能力 具有不同氧化还原能力的相关细胞色素的敏感氢交换变化 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.