SITE-SPECIFIC PROPERTIES OF A UNIQUE IRON-SULFUR PROTEIN

独特的铁硫蛋白的位点特异性

• 批准号: 2183276
• 负责人: Michael W. Adams
• 金额: $ 15.95万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183276
• 关键词: Archaea; Mossbauer spectrometry; Raman spectrometry; X ray crystallography; bacterial proteins; biophysics; calorimetry; chemical binding; circular dichroism; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; ferredoxin; iron sulfur protein; ligands; metalloproteins; mutant; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis; structural biology; thermostability

Clusters of non-heme iron and inorganic sulfur are ubiquitous electron carriers in fundamental life process such as respiration, photosynthesis, fermentation, and nitrogen fixation. However, the diversity of the biological roles for iron-sulfur clusters has only recently become apparent. In addition to being a major determinant of protein structure and stability, functional roles for iron-sulfur clusters now include substrate binding and activation in numerous redox and non-redox enzymes, generation and/or stabilization of radical intermediates, control of gene expression in iron regulation, and electron and/or iron storage. The majority of these roles require site-specific reactions at iron-sulfur clusters. The overall objective of this research program is to understand the diverse functions and properties of iron-sulfur clusters at the molecular level by studying a simple iron-sulfur protein, the ferredoxin from the hyperthermophilic archaeon, Pyococcus furious, and organism that grows optimally at 100 C. P. furious ferredoxin (M=7,500) contains a single [Fe4S4] cluster. It is remarkable both for this extreme thermal stability (at least 12 hours at 95 C) in being the only example of 4Fe-ferredoxin that has non-cysteinyl ligation at a specific iron site, and in the ease of quantitative removal of this iron to give a [Fe3S4] cluster. This not only facilitates the formation of heterometallic clusters, [MFe3S4], where M is a first row transition metal it also allows investigations into site-specific chemistry at both homometallic and heterometallic iron-sulfur clusters in a biological environment. Moreover, it is the only 4Fe-ferredoxin whose gene has been cloned and successfully expressed at the holoprotein in Escherichia coli. A comprehensive research program is proposed involving the use of biophysical techniques to investigate native and site-directed mutant forms of P. furious ferredoxin. The combination of nuclear magnetic resonance, X-ray crystallography, and calorimetry will be used to determine protein structure and stability. The structural, redox, and electronic properties of the iron-sulfur center will be assessed using a range of complimentary techniques; nuclear magnetic resonance, X-ray crystallography, electron paramagnetic resonance, UV/visible/near-IR absorption and natural and magnetically-induced circular dichroism, resonance Raman, Mossbauer, electron nuclear double resonance, and x-ray absorption. The results will provide unique insights into the factors that determine protein "hyperthermostability" and the structural and functional diversity of biological iron-sulfur clusters.

非血红素铁和无机硫的簇是无处不在的电子 基本生活过程中的载体，例如呼吸，光合作用， 发酵和氮固定。 但是，多样性 铁硫簇的生物学作用直到最近才成为 显而易见。 除了是蛋白质结构的主要决定因素 和稳定性，铁硫簇的功能作用现在包括 许多氧化还原和非雷多斯酶的底物结合和激活， 自由基中间体的产生和/或稳定基因的控制 铁调节和电子和/或铁存储中的表达。 这 这些角色中的大多数都需要在铁 - 硫酸盐时进行特定于现场的反应 集群。 该研究计划的总体目标是 了解铁硫簇的各种功能和特性 在分子水平上，通过研究简单的铁硫蛋白， 超疗法的高氧还蛋白，狂暴的狂热和 生长在100 C时最佳生长的生物体 P. Furious Ferredoxin（M = 7,500）包含一个[FE4S4]簇。这是 对于这种极端的热稳定性而引人注目（至少12小时 95 c）是具有非胞晶的4FE-毒素的唯一例子 在特定铁地点的连接，并易于定量去除 该铁的[Fe3S4]簇。 这不仅有助于 杂质簇的形成，[MFE3S4]，其中m是第一行 过渡金属还允许对特定地点的调查 同层和异含铁硫簇的化学 在生物环境中。 而且，它是唯一的4FE-毒素 其基因已被克隆并成功地表达在多蛋白上 在大肠杆菌中。 提出了一项全面的研究计划 涉及使用生物物理技术研究天然和 愤怒的铁氧还蛋白的位置定向突变形式。 结合 核磁共振，X射线晶体学和量热法将会 用于确定蛋白质结构和稳定性。 结构， 氧化还原和铁硫心中心的电子特性将是 使用一系列免费技术评估；核磁 共振，X射线晶体学，电子顺磁共振，， 紫外线/可见/近红外吸收以及天然和磁性诱导的 圆二色性，共鸣拉曼，莫斯鲍尔，电子核双重双 共振和X射线吸收。 结果将提供独特的 深入了解确定蛋白质“高度突变性”的因素 以及生物铁硫的结构和功能多样性 集群。