STRUCTURAL STUDIES OF COMPLEX IRON-SULFUR FLAVOPROTEINS

复杂铁硫黄素蛋白的结构研究

• 批准号: 3279762
• 负责人: F SCOTT MATHEWS
• 金额: $ 17.5万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279762
• 关键词: X ray crystallography; adenosine diphosphate; computer graphics /printing; electron density; electron transport; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; flavin mononucleotide; flavoproteins; iron; metalloenzyme; molecular cloning; oxidoreductase; point mutation; protein engineering; protein sequence; sulfur compounds

The molecular structure of trimethylamine dehydrogenase (TMADH) will be completed at 2.4A resolution and extended to 1.8A resolution. Investigation of the catalytic mechanism will be carried out by difference Fourier studies of crystals modified by substitution and by site-directed mutagenesis. Finally, crystals of the electron transfer flavoprotein (ETF) and its complex with TMADH will be prepared and analyzed. The structure of TMADH from the methylotrophic bacterium W3A1 has been solved at 2.4A resolution and interpreted with an amino acid sequence derived from the electron density map. The protein is a symmetric dimer of Mr 166,000 with each subunit containing a covalently bound FMN, a (4Fe-4S) center and a molecule of ADP. The subunits each contain 3 domains. One domain is a beta 8 alpha 8 parallel beta barrel and contains the FMN and iron-sulfur center. The other two domains contain 5- stranded parallel alpha/beta structures, similar to glutathione reductase, with the ADP moiety lying between them. The DNA sequence of the TMADH gene, which is presently being cloned, will be determined and used to complete the 2.4A structure analysis. The data will then be extended to 1.8A resolution and used for refinement of the structure. Crystals will be studied in various redox states and with substrates and inhibitors bound to them at 2.4A resolution, in order to investigate the mechanism of enzyme action and intramolecular electron transfer. Site-specific mutagenesis of the cloned gene will also be carried out to study the structural and catalytic role of various amino acids. The ETF which serves as the natural electron acceptor for TMADH is a heterodimer of Mr 75,000 containing a single FAD cofactor. Crystals of ETF and its complex with TMADH will be prepared using techniques such as vapor diffusion, microdialysis or free-interface diffusion. The crystals will be analyzed by the multiple isomorphous replacement method and by computer graphics and refinement techniques.

三甲胺脱氢酶的分子结构 (TMADH)将以2.4A分辨率完成，并扩展到 1.8A分辨率。对催化机理的研究将在 用差分傅里叶方法研究了经 替换和定点突变。最后，晶体 电子转移黄素蛋白(ETF)及其与蛋白质的络合物 TMADH将进行准备和分析。 甲基营养细菌TMADH的结构 W3A1已在2.4A分辨率下解决，并使用 根据电子密度图推导出氨基酸序列。这个 蛋白质是Mr 166,000的对称二聚体，每个亚基 含有共价结合的FMN、(4Fe-4S)中心和 ADP分子。每个亚基包含3个结构域。一 域是一个Beta 8 Alpha 8并行测试桶，包含 FMN和铁硫中心。另外两个域包含5- 类似于谷胱甘肽的链状平行的α/β结构 还原酶，ADP部分位于两者之间。 TMADH基因的DNA序列，目前正在 克隆，将被确定并用于完成2.4A 结构分析。然后数据将扩展到1.8A 分辨率，并用于结构的改进。水晶将会 在不同的氧化还原状态下和底物和 抑制剂以2.4A的分辨率结合到它们上，以便 探讨酶的作用机制和分子内机制 电子转移。克隆基因的定点突变 还将开展结构和催化作用的研究 各种氨基酸的结合。 作为天然电子受体的ETF TMADH是一种含有单一FAD的mR为75000的杂二聚体 辅因。ETF及其与TMADH的络合物的晶体将被 使用蒸气扩散、微渗析或 自由界面扩散。这些晶体将由 一种多重同构置换方法及计算机 图形和精细化技术。