Structure/Function Modularity in Nitric Oxide Synthase

描述(申请人提供)：本研究计划的主要目标是确定结构参数，这些参数定义了一氧化氮合酶(NOS)各亚型在不同环境中的功能。L-精氨酸是一氧化氮合酶的单一天然底物，产生L-瓜氨酸和一氧化氮，一氧化氮在影响神经传递、细胞毒性或血管扩张等生物效应中起到气体信使的作用。有三个基因编码一氧化氮合酶蛋白：神经型一氧化氮合酶(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(NOS3；eNOS)以及其他一些基因产物，这些基因产物存在于各种组织中，是RNA选择性剪接的结果。所有的一氧化氮合酶亚型都需要NADPH作为L-精氨酸氧化的还原当量的来源，以形成NO。一氧化氮合酶亚型的基本化学机制与细胞色素P450介导的反应相似，但不同亚型之间电子等价物对代谢产物产生的偶联程度、总反应速度和催化活性的调节明显不同。通过了解它们各自的结构属性，可以设计特定的化学干预措施来调节每种异构体的活动。总体假设是，尽管需要相同的辅基和辅因子(FAD、FMN、Fe-原卟啉IX、锌和四氢生物蝶呤)来催化相同的酶反应，但NOS亚型已经进化出不同的序列和结构属性来适应它们不同的功能。其具体目标是：1)使用结晶学方法继续在原子水平上研究结构性质，并使用分析超速离心法、电子显微镜和高压光谱分析来确定NOS全酶和衍生结构域的其他生物物理性质；2)利用包括共结晶和表面等离子体共振技术在内的各种生物物理方法来表征调节NOS活性的蛋白质-蛋白质相互作用，以测量与缓激肽受体、小窝蛋白、动力素和nostrin或它们各自的相互作用结构域的相互作用；以及3)鉴定和量化在各种条件下由NOS亚型产生的各种还原氧物种(O2-、H202、Oono-)，包括与蛋白质调节因子的相互作用，例如细胞环境中的那些调节因子。结合定点突变体、嵌合体和模块化结构来解剖和描述这些过程，已经在这个实验室成功地进行了这样的研究。

DESCRIPTION (provided by applicant): The principal objective of this research program is to determine the structural parameters that define the functions of the isoforms of nitric oxide synthase (NOS) in their various milieus. L-Arginine is the single natural substrate for the NOS enzymes, producing both L-citrulline and NO., which serves as a gaseous messenger in effecting neurotransmission, cytotoxicity, or vasodilatation, among other biological effects. Three genes encode the NOS proteins: neuronal NOS (NOS-1; nNOS), inducible NOS (NOS-2; iNOS), and endothelial NOS (NOS 3; eNOS) and a number of other gene products found in various tissues resulting from alternative RNA splicing. All NOS isoforms require NADPH as a source of reducing equivalents for oxygenation of L-arginine to form NO.. The basic chemical mechanisms of NOS isoforms are similar to those demonstrated for cytochrome P450- mediated reactions but the extent of coupling of electron equivalents to the production of metabolites, overall reaction rates, and regulation of catalytic activity vary significantly among the isoforms. By understanding their individual structural properties, specific chemical interventions can be designed to regulate the activities of each of the isoforms. The overall hypothesis is that, despite requiring the identical complement of prosthetic groups and cofactors (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin) to catalyze the same enzymatic reaction, NOS isoforms have evolved different sequences and structural properties to accommodate their distinct functions. The Specific Aims are: 1) to continue examining structural properties at the atomic level, using crystallographic methods, and to determine other biophysical properties of the NOS holoenzymes and derivative domains, using analytical ultracentrifugation, electron microscopy, and high pressure spectroscopy that determine their unique characteristics; 2) to characterize the protein-protein interactions that regulate NOS activities, using various biophysical methods including co-crystallization and surface plasmon resonance techniques, to measure interactions with bradykinin receptors, caveolins, dynamin, and nostrin or their respective interactive domains; and 3) to identify and quantify the various reduced oxygen species (O2-, H202, OONO-) produced by the NOS isoforms under a variety of conditions, including interactions with protein regulators, such as those in the cellular environment. A combination of site-directed mutants, chimeras, and modular constructs to dissect and characterize these processes has been used successfully in performing such studies in this laboratory.