STRUCTURAL/FUNCTIONAL MODULARITY IN NITRIC OXIDE SYNTHAS

描述：(逐字摘自申请者摘要)总体目标， 关于一氧化氮合酶(NOS)的结构参数 确定功能，与最初的项目期没有变化。范围 这项研究项目的发展很大，包括了这三个 一氧化氮合酶的异构体(神经元型一氧化氮合酶(NOS-1：nNOS)、诱导型一氧化氮合酶(NOS-2；iNOS)和 内皮型一氧化氮合酶(NOS-2；eNOS))，每一种都利用还原等价物 从NADPH经两次连续氧化生成L-瓜氨酸和NO 台阶。尽管异构体的基本化学机制相似， 其减量当量与生产的耦合程度 含氧产物、总反应速率和调节不同 意义重大。这些差异与这些基因的生物学作用密切相关 神经元信号传递中的酶，对细菌攻击的免疫反应的控制， 和调节血管扩张。确定它们的结构是很重要的 各种异构体的性质，以便开发方法 通过治疗来不同地调节这些酶的活性 干预。它假设，尽管它们在要求 相同的假体基团和辅因子(FAD、FMN、Fe-原卟啉IX和 四氢生物蛋白)来催化相同的酶反应、序列和 一氧化氮合酶亚型的结构差异已经演变为适应它们的 单独的功能。 具体目标是根据两个主要的蛋白质结构域进行组织的 所有三种同工酶：1)血红素结合(加氧酶)和2)黄素结合 (还原酶)结构域。具体目标1：展示结构/功能 ALL加氧酶(血红素结合)结构域二聚化的意义 一氧化氮合酶的三种异构体，决定了金属中心的作用，由 由PI和合作者进行的硫化锌的结晶学和生化研究， 并确定在调节一氧化氮合酶中重要的蛋白质-蛋白质相互作用部位 功能。方法包括定点/缺失突变、化学 用于结晶学和生物化学的新型一氧化氮合酶构造物的研究进展 相互作用的细胞成分的鉴定。具体目标2：确定 黄素结合区功能的结构决定因素 控制该区域内和区域之间电子流动的三种一氧化氮合酶异构体 黄素蛋白和加氧酶结构域的定点定位和缺失分析 诱变、交联实验和荧光化肽的测量 蛋白质分子内和分子间的相互作用。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The overall goal, regarding the structural parameters of nitric oxide synthase (NOS) that determine function, has not changed from the initial project period. The scope of this research project has developed significantly to include all three isoforms of NOS (neuronal NOS (NOS-1: nNOS), inducible NOS (NOS-2; iNOS), and endothelial NOS (NOS-2; eNOS)), each of which utilizes reducing equivalents from NADPH to form L-citrulline and NO through two successive oxygenation steps. Although the basic chemical mechanisms of the isoforms are similar, their extent of coupling of reducing equivalents to the production of oxygenated products, overall reaction rate, and regulation differ significantly. These differences are germane to the biological roles of these enzymes in neuronal signaling, control of immune responses to bacterial insult, and regulation of vasodilatation. It is important to determine their structural properties of the individual isoforms in order to develop methods for differentially regulating the activities of these enzymes through therapeutic intervention. It is hypothesized that, despite their similarities in requiring the same prosthetic groups and cofactors (FAD, FMN, Fe-protoporphyrin IX, and tetrahydrobioptein) to catalyze the same enzymatic reaction, sequence and structural differences have evolved in NOS isoforms to accommodate their individual functions. The specific aims are organized according to the two major protein domains shared by all three isoforms: 1) Heme-binding (Oxygenase) and 2) Flavin-binding (Reductase) domains. Specific Aim 1: Demonstrate the structural/functional significance of the dimerization of the oxygenase (heme-binding) domains of all three isoforms of NOS, determine the role of the metal center, identified by crystallography and biochemical studies as ZnS4 by the PI and collaborators, and identify protein-protein interaction sites important in regulating NOS function. Methods include site-directed/deletion mutagenesis, chemical cross-linking, and development of novel NOS constructs for crystallography and identification of interacting cellular components. Specific Aim 2: Ascertain the structural determinants of the function of the flavin-binding domain of the three NOS isoforms in controlling electron flow within this domain and between the flavoprotein and oxygenase domains using site-directed and deletion mutagenesis, cross-linking experiments, and fluoresceinated peptides to measure intra- and intermolecular protein-protein interactions.