Structure and Dynamics of Ferredoxins by NMR and X-ray

通过 NMR 和 X 射线测定铁氧还蛋白的结构和动力学

• 批准号: 9215142
• 负责人: John Markley
• 金额: $ 20万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9215142&HistoricalAwards=false
• 关键词: Structure; Dynamics; Ferredoxins; NMR; ray

Two-iron ((2Fe-2S)) ferredoxins comprise an important class of biological molecules whose function is incompletely understood at the molecular level. The investigators propose to study three classes of (2Fe-2S) ferredoxins: those found in mitochondria of steroid metabolizing tissue in vertebrates, those involved in photosynthesis, and those involved in nitrogen fixation. Examples of wild-type ferrodoxins from each of the three categories have been cloned and currently are overproduced in the PI's laboratory. The investigators previously determined x-ray structures from examples of two of the ferredoxin classes and an NMR structure from one. NMR and x-ray studies of members of the other classes of (2Fe-2S) ferredoxin are underway. The experiments are designed (1) to develop an understanding of factors that determine the reduction potential and electron delocalization in (2Fe-2S) ferredoxins, (2) to elucidate mechanisms of electron transfer to and from these ferredoxins, and (3) to investigate the requirements and energetics of (2Fe-2S) cluster formation. Roles of individual residues will be investigated by site-directed mutagenesis. Stable isotope labels inserted at specific locations will be used to monitor the electronic properties of atoms in the active site. X-ray structures will be determined for interesting mutant proteins that can by crystallized and analyzed either by multiple isomorphous replacement or by molecular replacement methods. %%% One of the basic motifs found in proteins that function as electron carriers is an iron atom coordinated by four sulfur atoms (an iron- sulfur cluster). Iron-sulfur clusters are found in all forms of life from bacteria to man. Iron-sulfur clusters can contain one, two, three, four, or more iron atoms, and their affinity for electrons varies over several orders of magnitude. They are involved in photosynthesis, respiration, nitrogen fixation, and many other important metabolic processes. This research project is designed to answer the following fundamental questions about proteins that contain iron-sulfur clusters: (1) How does the sequence of the protein specify the structure of the iron-sulfur cluster, the number of iron atoms and the geometry? (2) How does the sequence of the protein determine the affinity of the cluster for electrons and thus tune its reactivity for a particular process? (3) What residues in the protein are responsible for the specificity of electron transfer, and how do they guide that transfer? This knowledge will enable one to design electron transfer proteins with desired properties.

双铁（（2Fe-2S））铁氧化还原蛋白包括一类重要的铁氧化还原酶。 生物分子，其功能尚不完全清楚， 分子水平。 研究人员建议研究三个 （2Fe-2S）铁氧化还原蛋白类：在线粒体中发现的那些， 脊椎动物的类固醇代谢组织， 光合作用，以及那些参与固氮。 示例 来自这三类中的每一类的野生型铁氧蛋白 已经被克隆，目前在PI的实验室里生产过剩。 研究人员先前确定了X射线结构， 两种铁氧还蛋白类的实例和来自 一个. 其他类别的成员的NMR和X射线研究 （2Fe-2S）铁氧还蛋白。 实验设计（1） 了解决定减少的因素 （2Fe-2S）铁氧化还原蛋白中的电位和电子离域，（2） 为了阐明电子转移的机制， 铁氧还蛋白，和（3）调查的要求和能量学 （2Fe-2S）团簇的形成。 单个残基的作用将 通过定点诱变进行研究。 稳定同位素 在特定位置插入的标签将用于监视 活性部位原子的电子性质。 x射线 将确定感兴趣的突变蛋白的结构， 可以通过结晶和分析，或者通过多个同晶 置换或通过分子置换方法。 %%% 在蛋白质中发现的一种基本图案， 载体是由四个硫原子配位的铁原子（铁， 硫簇合物）。 铁硫团簇存在于各种形式的 从细菌到人类。铁硫簇可以包含一个， 两个、三个、四个或更多个铁原子，以及它们对 电子在几个数量级上变化。 他们是 参与光合作用、呼吸作用、固氮作用， 许多其他重要的代谢过程。 该研究项目是 旨在回答以下基本问题， 含有铁硫簇的蛋白质：（1） 蛋白质的序列指定了铁硫的结构 团簇，铁原子的数量和几何形状？(2)如何 蛋白质的序列决定了簇的亲和力 从而调整它的反应性， 流程？(3)蛋白质中的哪些残基负责 电子转移的特异性，以及它们如何引导电子转移， 转移？这些知识将使人们能够设计电子 具有所需性质的转移蛋白。