Structural/Functional Modularity in Nitric Oxide Synthase

描述（由申请人提供）：本研究计划侧重于控制一氧化氮合酶（NOS）异构体，神经元NOS (nNOS)，诱导NOS （iNOS）和内皮NOS （eNOS）在各自环境中的功能行为的结构特性。三个基因编码NOS酶，这些基因的其他产物在各种组织中作为选择性RNA剪接的结果表达。l -精氨酸是NOS异构体的天然底物，产生l -瓜氨酸和NO，根据异构体和组织定位，在神经传递、细胞毒性或血管扩张等过程中作为气态信使。本文提出的假设是，NOS异构体的分子设计虽然需要相同的辅因子和辅基（FAD、FMN、Fe-protoporphyrin IX、Zn和tetrahydrobiopterin），但在每个异构体中都进行了调整，以满足其特定的细胞功能。例如，在NOS组成酶（nNOS和eNOS）中插入的序列赋予了诱导异构体（iNOS）中不存在的调节特性。因此，分子研究将集中在进一步研究结构特性，利用新技术研究内在调控元件和关系，并确定影响氧代谢的机制。具体目的1：利用晶体学和低温电子显微镜确定NOS全酶及其衍生物结构域的结构性质；具体目标2：通过核磁共振波谱、激光闪光光解和蛋白膜伏安法研究nNOS和eNOS的内在调控，以确定这些蛋白的机制性质；具体目标3：利用快速冻灭ENDOR和传统的氧代谢测量方法，研究氧代谢和氧合过程的机制。这些实验将验证以下假设：1)每个NOS异构体的整体结构决定了这些酶对电子转移的内在控制；2)这些同工异构体的结构特征影响了它们的催化调节；3)不同的氧代谢也基于三种同工异构体的结构特征。公共卫生相关性：一氧化氮合酶产生的一氧化氮在人类神经传递、血液动力学控制和免疫反应中起着至关重要的作用。一氧化氮的异常产生与内皮功能障碍、感染性休克和其他疾病过程有关。在体外研究NOS的结构和调控将有助于更好地理解并积极影响人类的这些过程。

DESCRIPTION (provided by applicant): This research proposal focuses on structural properties that govern the functional behavior of the nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in their respective environments. Three genes encode NOS enzymes, and other products of these genes are expressed in various tissues as a result of alternative RNA splicing. L-Arginine is the natural substrate for NOS isoforms, producing L-citrulline and NO, which serves as a gaseous messenger in the processes of neurotransmission, cytotoxicity or vasodilatation, among others, depending upon the isoform and tissue localization. The hypotheses to be addressed in this proposal are that the molecular design of the NOS isoforms, while requiring the same cofactors and prosthetic groups (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin), is adapted in each isoform to satisfy its specific cellular function. For example, sequence inserts in the constitutive NOS enzymes (nNOS and eNOS) confer regulatory properties that do not exist in the inducible isoform (iNOS). Therefore, molecular studies will be focused on further examination of structural properties, using new techniques to examine intrinsic regulatory elements and relationships and the determination of mechanisms that bear on O2 metabolism. Specific Aim 1: To determine the structural properties of the NOS holoenzymes and derivative domains, using crystallography and cryo-electron microscopy; Specific Aim 2: To address intrinsic regulation of the nNOS and eNOS by nuclear magnetic resonance spectroscopy, laser flash photolysis and protein film voltammetry to determine the mechanistic properties of these proteins; and Specific Aim 3: To address the mechanisms involved in O2 metabolism and the process of oxygenation, using rapid-freeze-quench ENDOR, and conventional O2 metabolism measurements. These experiments will test the following hypotheses: 1) that the overall architecture of each NOS isoform determines the intrinsic control of electron transfer through these enzymes; 2) that these isoform-specific architectural features influence their catalytic regulation; and 3) that differential O2 metabolism is also based on structural features of the three isoforms. PUBLIC HEALTH RELEVANCE: The nitric oxide produced by the nitric oxide synthases plays vital roles in human neurotransmission, hemodynamic control, and the immune response. Aberrant production of nitric oxide has been implicated in endothelial dysfunction, septic shock, and other disease processes. Studying the structure and regulation of NOS in vitro will lead to greater the understanding of, and the ability to positively affect, these processes in humans.