TWO-DIMENSIONAL NMR STUDIES OF CYTOCHROME C FOLDING

这个项目的长期目标是更好地了解 蛋白质折叠反应中涉及的力和相互作用 细胞色素c作为模型蛋白。理解这一问题的一个主要障碍 蛋白质折叠过程一直是获得结构的难点 关于部分折叠的中间状态的数据。氢交换标记 与本实验室合作开发的快速混合方法 二维核磁共振波谱使观察地层成为可能 在复性过程中氢键结构的变化。细胞色素c的研究进展 和其他蛋白质已经表明，这种方法提供了空间和 时间分辨率，以获得详细的结构和动力学描述 可折叠的小路。 进一步了解细胞色素c的完整机制 折叠包括以下内容：(1)折叠中间体的稳定性 早期折叠事件将通过H-交换标记研究进行探索 在不同的复性和标记条件下；(2)血红素的作用 将探索折叠过程中的连接和脯氨酸异构化 野生型和突变型细胞色素的结构和动力学研究 (3)利用圆二色谱和二维核磁共振对合成的化合物进行表征 细胞色素c来源的多肽和蛋白水解物片段的搜索 用于螺旋结构和螺旋配对反应；(4) 细胞色素c折叠中的单个残基和相互作用将是 通过将结构方法与场地定向相结合进行探索 诱变。其他计划包括对细菌的折叠研究 细胞色素c与细胞色素C络合物的细胞色素和氢交换研究 单抗在寻找抗体诱导构象中的作用 改变。 它们提供了对蛋白质折叠的更好的结构理解 实验研究将对几个基础和应用具有重要意义 研究领域。这些努力包括从理论上破译 氨基酸序列编码的结构信息与生物技术 产品设计。

The long-term objective of this project is a better understanding of the forces and interactions involved in protein folding reactions using cytochrome c as a model protein. A major hurdle in understanding the process of protein folding has been the difficulty of obtaining structural data on partially folded intermediate states. Hydrogen exchange labeling and rapid mixing methods developed in this laboratory in conjunction with two-dimensional NMR spectroscopy make it possible to observe the formation of H-bonded structure during refolding. Previous results on cytochrome c and other proteins have shown that this approach provides the spatial and temporal resolution to obtain a detailed structural and kinetic description of folding pathways. Further steps towards a complete mechanistic understanding of cytochrome c folding include the following: (1) the stability of folding intermediates and early folding events will be probed by H-exchange labeling studies under various refolding and labeling conditions; (2) the role of heme ligation and proline isomerization in folding will be explored by structural and kinetic studies on wild-type and mutant forms of cytochrome c; (3) circular dichroism and 2D NMR will be used to characterize synthetic peptides and proteolytic fragments derived from cytochrome c in a search for helical structure and helix-pairing reactions; (4) the importance of individual residues and interactions in cytochrome c folding will be explored by combining the structural approaches with site-directed mutagenesis. Additional plans include folding studies on bacterial cytochromes and H-exchange studies on the complex of cytochrome c with monoclonal antibodies in a search for antibody-induced conformational changes. The better structural understanding of protein folding provided by these experimental studies will be important for several basic and applied research areas. These include theoretical efforts to decipher the structural information encoded in amino acid sequences and biotechnology product design.