Structural/Functional Modularity in Nitric Oxide Synthase

描述（由申请人提供）：本研究计划的重点是控制一氧化氮合酶（NOS）亚型、神经元NOS（nNOS）、诱导型NOS（iNOS）和内皮型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作为选择性RNA剪接的结果在各种组织中表达。L-精氨酸是NOS亚型的天然底物，产生L-瓜氨酸和NO，在神经传递、细胞毒性或血管舒张等过程中作为气体信使，这取决于亚型和组织定位。在这个建议中要解决的假设是，NOS亚型的分子设计，同时需要相同的辅因子和辅基（FAD，FMN，铁-原卟啉IX，锌和四氢生物蝶呤），是适应于每个亚型，以满足其特定的细胞功能。例如，组成型NOS酶（nNOS和eNOS）中的序列插入物赋予诱导型同种型（iNOS）中不存在的调节特性。因此，分子研究将集中在结构特性的进一步检查，使用新技术来检查内在的调节元件和关系，并确定承担O2代谢的机制。具体目标1：使用晶体学和冷冻电子显微镜确定NOS全酶和衍生结构域的结构特性;具体目标2：通过核磁共振光谱、激光闪光光解和蛋白膜伏安法解决nNOS和eNOS的内在调节，以确定这些蛋白质的机械特性;以及具体目标3：使用快速冷冻淬灭ENDOR和常规O2代谢测量来解决O2代谢和氧合过程中涉及的机制。这些实验将测试以下假设：1）每个NOS亚型的整体结构决定了通过这些酶的电子转移的内在控制; 2）这些亚型特异性结构特征影响其催化调节; 3）差异O2代谢也基于三种亚型的结构特征。 公共卫生相关性：由一氧化氮合酶产生的一氧化氮在人体神经传递、血流动力学控制和免疫反应中起着重要作用。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。研究体外NOS的结构和调节将有助于更好地理解和积极影响人类这些过程的能力。

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.