STRUCTURE, FUNCTION, & DYNAMICS OF P-450 CYTOCHROMES

结构、功能、

• 批准号: 3466229
• 负责人: SHAUN BLACK
• 金额: $ 10.05万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466229
• 关键词: NADPH cytochrome c2 reductase; X ray crystallography; affinity chromatography; chemical structure function; circular dichroism; computer data analysis; conformation; cytochrome P450; cytochrome b5 reductase; enzyme linked immunosorbent assay; enzyme model; enzyme structure; hemoprotein structure; high performance liquid chromatography; isozymes; laboratory rabbit; membrane proteins; membrane structure; microsomes; molecular site; monoclonal antibody; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein sequence; synthetic peptide

Structure, function, and conformational dynamics of the cytochrome P-450 monooxygenase system are to be studied in the proposed research. (1) Topology of membrane-bound rabbit microsomal isozymes 2 and 4 will be probed via polyclonal and monoclonal antibodies as well as by specific and non-specific proteolysis. Immobilized polyclonal antibodies will be used to purify specific peptides released upon protease treatment of microsomes; HPLC-isolated peptides will be submitted to micro- sequence analysis to reveal sites within the primary structures available at the external (cytoplasmic) surface of the endoplasmic reticulum. A panel of monoclonal antibodies will be raised to synthetic peptides selected from each sequence by MSEQ computer analysis; these site-specific antibodies will be utilized to probe native and protease-digested microsomes via ELISA and Western-blotting procedures. Together, the results obtained will permit reconstruction of the membrane topological features of each cytochrome. Preliminary data and calculations suggest, in contrast to current theory, that the topology of the P-450's is simple and is similar to that known for other microsomal proteins. Isozymes 2 and 4 were chosen for the proposed studies as they are the major forms present in phenobarbital-induced (isozyme 2) and aryl hydrocarbon-induced (isozyme 4) animals, they represent two major gene sub-families, they typify low- and high-spin cytochrome types, they are involved in drug disposition tolerance, and they participate in the oxidative metabolism of physiological lipids, numerous drugs, and other xenobiotics; furthermore, they are highly similar to forms known in the human. (2) Conformational dynamics of isozyme 2 will be studied with site- specific chemical modification of Cys-152, a residue shown to serve as a "reporter" for binding events at the active site. Modification kinetics, carbon-13 NMR spectroscopy, and circular dichroism will be utilized to examine the role and importance of conformational changes in the function of the cytochrome. Functional aspects to be examined will include substrate binding, heme reduction kinetics, and protein-protein interactions. (3) Crystallization of many of the microsomal monooxygenase system components will be attempted with the ultimate aim of three- dimensional structure elucidation. Exploration of the "small Amphiphile" concept and co-crystallization will be major thrusts. A long-term objective of these studies is to achieve a sophisticated molecular view of the P-450 system that will serve to guide rational drug design.

的结构、功能和构象动力学 细胞色素P-450单加氧酶系统将在 拟开展的研究。(1)膜结合型兔的拓扑结构 微粒体同工酶2和4将通过多克隆和 单抗以及特异性和非特异性 蛋白质分解。固定化多克隆抗体将用于 蛋白水解酶释放的特异性多肽的提纯 微粒体；高效液相分离的多肽将提交给微型- 序列分析以揭示初级结构中的位置 在内质的外(细胞质)表面可用 网状结构。一组克隆抗体将被提升到 MSEQ从每个序列中选择合成肽 计算机分析；这些特定部位的抗体将被利用 用酶联免疫吸附试验检测天然和酶消化的微生物体 Western-blotting程序。总而言之，取得的成果将 允许重建膜的拓扑特征 每种细胞色素。初步数据和计算表明，在 与目前的理论相反，P-450型S的拓扑结构是 简单，与已知的其他微体蛋白相似。 同工酶2和4被选为拟议研究的原样 苯巴比妥诱导的主要形式(同工酶2)和 芳烃诱导(同工酶4)动物，它们代表两个 主要基因亚家族，它们代表低和高自旋 细胞色素类型，它们与药物处置耐受有关， 它们参与生理性的氧化代谢 脂类、多种药物和其他外来生物；此外，他们 与人类已知的形式高度相似。(2) 同工酶2的构象动力学将用Site-Site来研究。 Cys-152的特定化学修饰，一个残基显示 担任活动现场约束性活动的“记者”。 改性动力学、~(13)C核磁共振波谱和环化反应 我们将利用二色性来考察 细胞色素功能的构象变化。 要检查的功能方面将包括底物结合， 血红素还原动力学和蛋白质-蛋白质相互作用。(3) 许多微粒体单加氧酶系统的结晶 将尝试组件，最终目标是三个- 空间结构的阐明。对“小”的探索 “两亲性”概念和共晶化将是主要的推动力。 这些研究的长期目标是实现 P-450系统的复杂分子视图将服务于 指导合理用药设计。