Structure/Function Modularity in Nitric Oxide Synthase

描述（由申请人提供）：本研究计划的主要目的是确定定义一氧化氮合酶（NOS）亚型在其各种环境中的功能的结构参数。L-精氨酸是NOS酶的单一天然底物，产生L-瓜氨酸和NO。其在影响神经传递、细胞毒性或血管舒张以及其它生物效应中充当气体信使。三种基因编码NOS蛋白：神经元NOS（NOS-1; nNOS）、诱导型NOS（NOS-2; iNOS）和内皮型NOS（NOS 3; eNOS），以及在各种组织中发现的由选择性RNA剪接产生的许多其他基因产物。所有NOS亚型都需要NADPH作为还原当量的来源，用于L-精氨酸的氧化以形成NO。NOS异构体的基本化学机制与细胞色素P450介导的反应类似，但电子等价物与代谢产物的产生、总体反应速率和催化活性调节的耦合程度在异构体中显著不同。通过了解它们各自的结构特性，可以设计特定的化学干预措施来调节每种亚型的活性。总体假设是，尽管需要相同的辅基和辅因子（FAD，FMN，铁-原卟啉IX，锌和四氢生物蝶呤）来催化相同的酶促反应，NOS同工型已经进化出不同的序列和结构特性，以适应其独特的功能。具体目标是：1）继续使用晶体学方法在原子水平上检查结构性质，并使用确定其独特特征的分析超离心、电子显微镜和高压光谱法来确定NOS全酶和衍生物结构域的其他生物物理性质; 2）表征调节NOS活性的蛋白质-蛋白质相互作用，使用各种生物物理方法，包括共结晶和表面等离子体共振技术，以测量与缓激肽受体、小窝蛋白、发动蛋白和nostrin或它们各自的相互作用结构域的相互作用;和3）鉴定和定量在各种条件下由NOS同种型产生的各种还原氧物质（O2-、H2 O2、OONO-），包括与蛋白质调节剂的相互作用，例如在细胞环境中的那些。定点突变体，嵌合体和模块化结构的组合，以解剖和表征这些过程已被成功地用于在本实验室进行这样的研究。

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.