STRUCTURAL STUDIES OF COMPLEX IRON-SULFUR FLAVOPROTEINS

复杂铁硫黄素蛋白的结构研究

• 批准号: 3279755
• 负责人: F SCOTT MATHEWS
• 金额: $ 19.97万
• 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 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279755
• 关键词: X ray crystallography; alcohol dehydrogenase; amine oxidoreductase; cytochrome c; enzyme mechanism; enzyme structure; enzyme substrate complex; flavin mononucleotide; flavoproteins; iron sulfur protein; metalloenzyme; methanol; quinones; site directed mutagenesis

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 催化机理的研究将是 进行了不同的傅立叶研究的晶体改性， 取代和通过定点诱变。 最后， 电子转移黄素蛋白（ETF）及其与 将制备并分析TMADH。 甲基营养菌TMADH的结构 W3 A1已在2.4A分辨率下求解，并使用 氨基酸序列来源于电子密度图。 的 蛋白质是对称的二聚体，每个亚基的Mr为166，000 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beta 8 alpha 8平行beta桶，包含 FMN和铁硫中心。 其他两个域包含5- 链平行α/β结构，类似于谷胱甘肽 在它们之间有ADP部分。 TMADH基因的DNA序列，这是目前正在研究的。 克隆，将被确定并用于完成2.4A 结构分析 然后将数据扩展到1.8A 分辨率，并用于结构的细化。 晶体将 在各种氧化还原状态下和与底物一起进行研究， 抑制剂以2.4A分辨率与它们结合，以便 研究酶的作用机制和分子内 电子转移 克隆基因的定点突变 还将进行结构和催化作用的研究 各种氨基酸。 ETF作为天然电子受体， TMADH是一种Mr为75，000的异二聚体，含有一个FAD 辅因子 ETF的晶体及其与TMADH的复合物将被 使用诸如蒸气扩散、微透析或 自由界面扩散 这些晶体将由 多同构置换法及其计算机实现 图形和细化技术。