STRUCTURAL/FUNCTIONAL MODULARITY IN NITRIC OXIDE SYNTHAS

描述：（申请人摘要中的一字不差）总体目标， 关于一氧化氮合酶（NOS）的结构参数， 确定功能，从最初的项目期间没有改变。范围 这个研究项目的发展有了很大的进步， NOS亚型（神经元型NOS（NOS-1：nNOS）、诱导型NOS（NOS-2; iNOS）和 内皮NOS（NOS-2; eNOS）），每一种都利用还原当量 通过两次连续氧化，从NADPH生成L-瓜氨酸和NO 步尽管同种型的基本化学机制相似， 还原当量与生产的耦合程度 含氧产物、总反应速率和调节不同 显著这些差异是密切相关的生物作用， 神经元信号传导中的酶，对细菌损伤的免疫反应的控制， 和调节血管舒张。重要的是要确定其结构 为了开发用于以下的方法， 差异调节这些酶的活性，通过治疗 干预据推测，尽管它们在要求上有相似之处， 相同的辅基和辅因子（FAD，FMN，Fe-原卟啉IX，和 四氢生物肽）催化相同的酶促反应，序列和 NOS同工型的结构差异已经进化，以适应它们的 个别功能。 具体的目标是根据两个主要的蛋白质结构域组织的 所有三种亚型共有：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 所有的加氧酶（血红素结合）结构域的二聚化的意义 NOS的三种亚型，决定金属中心的作用，通过 PI和合作者的晶体学和生物化学研究，如ZnS 4， 并鉴定在调节NOS中重要的蛋白质-蛋白质相互作用位点 功能方法包括定点/缺失诱变、化学诱变、酶联免疫吸附试验、酶联免疫吸附试验和酶联免疫吸附试验。 交联和开发用于晶体学的新型NOS构建体， 识别相互作用的细胞成分。具体目标2：确定 的黄素结合结构域的功能的结构决定因素， 三种NOS同工型在控制电子流在这个领域和之间 黄素蛋白和加氧酶结构域使用定点和缺失 诱变、交联实验和荧光素化肽来测量 分子内和分子间蛋白质-蛋白质相互作用。

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.