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: 3289368
• 负责人: HEINRICH RODER
• 金额: $ 20.57万
• 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/3289368
• 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.

这个项目的长期目标是更好地理解