Assembly of Biological Iron-Sulfur Clusters

描述(申请人提供)：铁-硫团簇存在于160多种不同类型的酶或蛋白质中，构成了最古老、最普遍和结构最多样化的生物假体基团之一。因此，铁-硫的生物合成过程对几乎所有形式的生命都是必不可少的，并且在原核生物和真核生物中非常保守。已经出现了三种不同类型的铁-硫簇组装机制，称为NIF、ISC和SUF系统，在每种情况下，总的机制都涉及半胱氨酸脱硫酶介导的临时簇在支架蛋白上的组装，以及随后预先形成的簇或簇片段转移到载脂蛋白。然而，在任何情况下，组装或转移机制都不是在分子水平上理解的。该项目的长期目标是在分子水平上了解NIF、ISC和SUF系统中铁硫簇合物的生物合成。阐明铁-S簇生物合成的机制对于理解细胞铁的动态平衡以及与铁超载和线粒体呼吸链缺陷相关的人类疾病具有重要意义。该方法包括使用分子生物学技术来实现目标酶和蛋白质的大规模表达和/或位点特异性变化，生化和酶分析，X射线结晶学，以及生物物理光谱技术(电子顺磁共振、电子吸收和磁圆二色谱、共振拉曼、穆斯堡尔和质谱学)的应用，这些技术可以探索团簇生物合成过程中铁或铁硫中心的性质和详细性质。本研究的目的是建立已知的三种铁硫簇支架蛋白的簇结合形式的结构和簇组装和转移的分子机制，表征铁硫簇生物合成的调控机制，并鉴定迄今表征的立方烷[4Fe-4S]簇组装所特有的支架蛋白。

DESCRIPTION (provided by applicant): Iron-sulfur clusters are present in more than 160 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of iron-sulfur biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged, termed the NIF, ISC and SUF systems, and in each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular-level understanding of iron-sulfur cluster biosynthesis in the NIF, ISC and SUF systems. Elucidating the mechanism of Fe-S cluster biosynthesis is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site specific changes in the target enzymes and proteins, biochemical and enzymatic assays, x-ray crystallography, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, electronic absorption and magnetic circular dichroism, resonance Raman, Mossbauer and mass spectrometry) that can probe the nature and detailed properties of iron or iron-sulfur centers during cluster biosynthesis. The objectives are to establish the structure of cluster-bound forms and the molecular mechanism of cluster assembly and transfer for each the three known types of iron-sulfur cluster scaffold proteins, characterize the mechanism of regulation of iron-sulfur cluster biosynthesis, and identify hitherto in characterized scaffold proteins specific of the assembly of cubane [4Fe-4S] clusters.