NMR SPECTROSCOPY OF IRON-SULFUR PROTEINS

铁硫蛋白的核磁共振波谱

• 批准号: 7340561
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 8.32万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7340561
• 关键词: Active Sites; Acyl Carrier Protein; Acyl-[acyl-carrier-protein] desaturase; Affect; Affinity; Anabolism; Arts; Biochemical Process; Biological Models; Chemicals; Class; Complex; Coupling; Data Collection; Defect; Drug Metabolic Detoxication; Electron Transport; Electronics; Electrons; Escherichia coli; Gene Expression Regulation; Goals; Grant; Hydrogen Bonding; Investigation; Iron; Iron-Sulfur Proteins; Label; Life; Ligands; Link; Metalloproteins; Metals; Mixed Function Oxygenases; Modeling; Molecular Chaperones; NMR Spectroscopy; Nature; Nuclear; Numbers; Oxidation-Reduction; Pathway interactions; Pattern; Play; Production; Property; Proteins; Protons; Range; Respiration; Roentgen Rays; Role; Rubredoxins; Spectrum Analysis; Staging; Structure; Structure-Activity Relationship; Sulfur; System; Technology; Thermus; Toluene; Work; X-Ray Crystallography; chemical property; human disease; insight; member; protein protein interaction; quantum; size

Iron-sulfur proteins are ubiquitous in nature and essential to life. Members of this class of metalloprotein play critical roles in biochemical processes ranging from respiration and detoxification to gene regulation and chemical sensing. Defects in iron-sulfur proteins are linked to a large number of human diseases. The electronic properties of iron-sulfur proteins, which impact on their function, are modulated by the geometry of their iron centers and by interactions between electrons on the metal(s) and the ligands provided by the protein. Our overall goals are to determine how protein-protein interactions facilitate iron-sulfur cluster assembly and modulate electron-nuclear interactions at the active sites of iron-sulfur proteins so as to facilitate electron transfers. Our experimental approach to understanding sequence-structure-function relationships in iron-sulfur proteins combines NMR spectroscopy and quantum chemical calculations with information from X-ray crystallography. NMR spectra contain exquisitely sensitive information about electron- nuclear interactions. This information, which is unavailable from X-ray crystal structures, provides insights into the chemical properties of the iron centers, details of their geometry, strengths of hydrogen bonds, proton affinities, and patterns of electron delocalization. NMR thus serves as a window for viewing the properties of the cluster that control redox potentials and regulate pathways of electron transfer and redox dependent changes in pKa values. In the past, we have applied this approach to investigations of single iron- containing proteins and proteins that interact with them. We propose here to extend these investigations to biologically important protein-protein complexes that involve iron centers. Our specific aims are:(1) to determine the mechanism of coupling between proton and electron transfer in the Rieske protein from Thermus thermophilis; (2) to determine how protein:protein complexes affect electron-nuclear interactions and to determine mechanisms of electron transfer in three model biological systems (rubredoxin:FNR, stearoyl-acyl carrier protein desaturase, and toluene monooxygenase system); (3) to determine mechanisms of chaperone-assisted iron-sulfur cluster assembly in Escherichia coli. To achive these aims, we will make use of state-of-the-art approaches to protein production and isotopic labeling, NMR data collection and analysis, modeling of structures and complexes, and quantum chemical calculations.

铁硫蛋白在自然界中普遍存在，对生命至关重要。此类金属蛋白的成员发挥作用 从呼吸和解毒到基因调控等生化过程中的关键作用 化学传感。铁硫蛋白的缺陷与许多人类疾病有关。这 铁硫蛋白的电子特性会影响其功能，并受到其几何形状的调节 它们的铁中心以及金属上的电子和由其提供的配体之间的相互作用 蛋白质。我们的总体目标是确定蛋白质-蛋白质相互作用如何促进铁-硫簇 在铁硫蛋白的活性位点组装并调节电子-核相互作用，从而 促进电子转移。我们理解序列-结构-功能的实验方法 铁硫蛋白的关系结合了核磁共振波谱和量子化学计算 来自 X 射线晶体学的信息。核磁共振谱包含有关电子的极其敏感的信息 核相互作用。这些无法从 X 射线晶体结构中获得的信息提供了见解 深入研究铁中心的化学性质、几何形状的细节、氢键的强度， 质子亲和力和电子离域模式。因此，NMR 可作为观察 控制氧化还原电位并调节电子转移和氧化还原途径的簇的特性 pKa 值的相关变化。过去，我们已将这种方法应用于单铁的研究 含有蛋白质和与其相互作用的蛋白质。我们在此建议将这些调查范围扩大到 涉及铁中心的生物学上重要的蛋白质-蛋白质复合物。我们的具体目标是：(1) 确定 Rieske 蛋白中质子和电子转移之间的耦合机制 嗜热栖热菌； (2) 确定蛋白质：蛋白质复合物如何影响电子-核相互作用 并确定三种模型生物系统中的电子转移机制（红氧还蛋白：FNR、 硬脂酰-酰基载体蛋白去饱和酶和甲苯单加氧酶系统）； (3)确定机制 大肠杆菌中分子伴侣辅助的铁硫簇组装。为了实现这些目标，我们将 使用最先进的方法进行蛋白质生产和同位素标记、NMR 数据收集和 分析、结构和复合物建模以及量子化学计算。