SITE DIRECTED MUTAGENESIS OF AZOTOBACTER FERREDOXIN I

固氮细菌铁氧化还原蛋白 I 的定点诱变

• 批准号: 3304588
• 负责人: BARBARA K BURGESS
• 金额: $ 35.53万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304588
• 关键词: Azotobacter; X ray crystallography; chemical synthesis; dichroism; electron nuclear double resonance spectroscopy; ferredoxin; gene mutation; nuclear magnetic resonance spectroscopy; peptides; protein purification; protein sequence; protein structure function; proteins; redoxin; site directed mutagenesis

The general goal of the work proposed here is to elucidate the interrelationships between protein structure and [Fe-S] cluster structure, function, redox properties and reactivity. To meet these goals we are using site-directed mutagenesis to modify the amino acid sequence of our model protein, Azotobacter vinelandii FdI. Following the expression of the mutated proteins in their native background they will be purified in large quantities and their properties will be completely characterized by X-ray crystallographic, biochemical, spectroscopic and electrochemical methods. The chemical behavior of these new forms of AvFdI, including their conversion to other so far unknown forms of AvFdI will also be characterized. In addition we will determine whether or not the new mutant forms of AvFdI are functional in vivo. This work constitutes the first application of site-directed mutagenesis to an [Fe-S] protein of known structure. The results will be applicable to the entire class of [Fe-S] proteins and can be expected to yield much new information relevant to the identification of [Fe-S] cluster structures in more "complex" [Fe-S] proteins yet to be characterized.

这里提出的工作的一般目标是阐明 蛋白质结构与[Fe-s]簇结构之间的相互关系， 功能，氧化还原特性和反应性。为了满足我们正在使用的目标 定向诱变以修改我们的模型的氨基酸序列 蛋白质，氮杂杆菌Vinelandii FDI。遵循表达 在其本地背景中突变的蛋白质将被大量纯化 数量及其特性将完全以X射线为特征 晶体学，生化，光谱和电化学方法。 这些新形式的AVFDI的化学行为，包括它们 到目前为止，avfdi的其他未知形式也将是 特征。另外，我们将确定新突变体是否 AVFDI的形式在体内功能性。这项工作构成了第一个 将定点诱变应用于已知的[Fe-S]蛋白 结构。结果将适用于[Fe-S]的整个类 蛋白质，可以期望产生与该蛋白质有关的许多新信息 在更“复杂” [fe-s]中识别[Fe-s]群集结构 蛋白质尚未表征。