HIGH-RESOLUTION CRYSTALLOGRAPHIC STUDIES OF TWO RESPIRATORY PROTEINS FROM T THE

两种呼吸蛋白的高分辨率晶体学研究

• 批准号: 7722120
• 负责人: JAMES A FEE
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722120
• 关键词: Binding; Biochemistry; Computer Retrieval of Information on Scientific Projects Database; Condition; Crystallization; Enzymes; Funding; Grant; Heme; Imidazole; In Situ; Institution; Ligation; Mutate; Mutation; Oxidases; Proteins; Protons; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Screening procedure; Source; Structure; System; Thermus; Time; United States National Institutes of Health; Work; cryogenics; cytochrome c oxidase; fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether; insight; interest; mutant; novel; oxidation; respiratory protein

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In order to garner powerful new insights on heme-Cu oxidase biochemistry, continued X-ray beam time is requested to perform crystallographic studies of the novel cytochrome c oxidase, ba3, from T. thermophilus. Our plans call for obtaining: (a) increased resolution of a new mutant form of the as-isolated, fully oxidized enzyme; (b) independent structures of numerous mutant forms notably: (i) of mutated proteins that crystallize in different space groups with the view of increasing reliability of crystallization in different oxidation and ligation states, and, we hope, through rational screening, will provide ~2.0 A resolution, (ii) to reveal both structure and function of the O2 delivery channel, (iii) to examine the structural effects of mutations in the so-called K-path of proton transport; (c) independent structures of the fully reduced enzyme followed by in situ oxygenation with gaseous O2 using a cryogenic flow system under conditions which permit formation of the cryo-stable A-compound, Fea3-O2. This constitutes the main objectives of our effort. However, we retain an interest in the Thermus Rieske protein. Previous studies yielded a 1.3 ¿ structure of the fully-oxidized Thermus Rieske protein at pH 8.5 in which one of the Fe-bound imidazole rings was deduced to be partially de-protonated. Our plans call for extending this work to include the following: (a) an atomic resolution (

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 为了获得关于血红素铜氧化酶生物化学的强大新见解，需要持续的 X 射线束时间来对来自嗜热链球菌的新型细胞色素 C 氧化酶 ba3 进行晶体学研究。我们的计划要求获得：（a）提高分离的完全氧化酶的新突变形式的分辨率； （b）许多突变形式的独立结构，特别是：（i）在不同空间群中结晶的突变蛋白，以提高不同氧化和连接状态下结晶的可靠性，并且我们希望通过合理筛选，将提供〜2.0 A的分辨率，（ii）揭示O2输送通道的结构和功能，（iii）检查所谓的质子运输K路径中突变的结构效应； (c) 完全还原的酶的独立结构，然后使用低温流动系统在允许形成低温稳定的 A-化合物 Fea3-O2 的条件下用气态 O2 进行原位氧化。这是我们努力的主要目标。然而，我们仍然对栖热菌 Rieske 蛋白感兴趣。先前的研究在 pH 8.5 下产生了完全氧化的 Thermus Rieske 蛋白的 1.3 ¿ 结构，其中 Fe 结合的咪唑环之一被推断为部分去质子化。我们的计划要求扩展这项工作，包括以下内容：(a) 原子分辨率（