TWO-DIMENSIONAL NMR STUDIES OF CYTOCHROME C FOLDING

细胞色素C折叠的二维核磁共振研究

• 批准号: 3289369
• 负责人: HEINRICH RODER
• 金额: $ 25.64万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289369
• 关键词: biochemical evolution; chemical models; circular dichroism; cis trans isomerization; conformation; cytochrome c; deuterium; hemoprotein structure; hydrogen bond; model design /development; monoclonal antibody; nonradiation isotope effect; nuclear magnetic resonance spectroscopy; nucleic acid sequence; proline; protein folding; protein sequence; protein structure; proteolysis; site directed mutagenesis; synthetic peptide

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.

该项目的长期目标是更好地了解 蛋白质折叠反应中的力和相互作用， 细胞色素c作为模型蛋白。 理解这一点的一个主要障碍是 蛋白质折叠的过程一直是获得结构的困难， 关于部分折叠中间状态的数据。 氢交换标记 本实验室开发的快速混合方法， 二维核磁共振光谱学使得有可能观察形成 重折叠过程中的氢键结构。 细胞色素c的先前结果 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 折叠路径。 细胞色素c完整机理的进一步研究 折叠的研究包括以下几个方面：（1）折叠中间体的稳定性 和早期折叠事件将探测的H-交换标记研究 在不同的重折叠和标记条件下;（2）血红素的作用 连接和脯氨酸异构化折叠将探讨 细胞色素野生型和突变型结构和动力学研究 （3）圆二色性和2D NMR将用于表征合成的 肽和蛋白水解片段来源于细胞色素c， 螺旋结构和螺旋配对反应;（4）的重要性， 细胞色素c折叠中的单个残基和相互作用将被 通过结合结构方法和现场指导 诱变 其他计划包括对细菌的折叠研究 细胞色素C与H2 O2复合物的细胞色素和H-交换研究 单克隆抗体用于寻找抗体诱导的构象 变化 这些研究提供了对蛋白质折叠的更好的结构理解 实验研究将是重要的几个基础和应用 研究领域。 这些包括理论上的努力，以破译 氨基酸序列编码的结构信息与生物技术 产品设计