Structure/Function Modularity in Nitric Oxide Synthase

描述（由申请人提供）：本研究项目的主要目的是确定确定一氧化氮合酶（NOS）同工型在不同环境下功能的结构参数。l -精氨酸是NOS酶的单一天然底物，可产生l -瓜氨酸和NO。，在影响神经传递、细胞毒性或血管扩张等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NOS (NOS-2; iNOS)，内皮NOS （NOS 3; eNOS）和其他一些基因产物，这些基因产物存在于各种组织中，由选择性RNA剪接产生。所有NOS异构体都需要NADPH作为l -精氨酸氧化形成NO的还原等效物的来源。NOS同工异构体的基本化学机制与细胞色素P450介导的反应相似，但电子当量与代谢物产生的耦合程度、总反应速率和催化活性的调节在不同的同工异构体之间差异很大。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同工异构体的活性。总体假设是，尽管需要相同的补体基团和辅因子（FAD、FMN、Fe-protoporphyrin IX、Zn和tetrahydrobiopterin）来催化相同的酶促反应，NOS同工异构体进化出不同的序列和结构性质以适应其不同的功能。具体目标是：1)继续在原子水平上研究结构特性，使用晶体学方法，并确定NOS全酶及其衍生物结构域的其他生物物理特性，使用分析性超离心，电子显微镜和高压光谱来确定其独特的特性；2)表征调节NOS活性的蛋白-蛋白相互作用，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，测量与缓激肽受体、小窝蛋白、动力蛋白和诺斯特宁或其各自的相互作用域的相互作用；3)鉴定和量化NOS同种异构体在各种条件下产生的各种还原氧（O2-, H202, OONO-），包括与蛋白质调节因子的相互作用，如细胞环境中的相互作用。位点导向突变体、嵌合体和模块化结构的组合来解剖和表征这些过程已成功地用于本实验室进行此类研究。

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.