RUI: Domain Shuttle Mechanisms for Catalysis and Control in Nitric Oxide Synthases

一氧化氮合酶产生一氧化氮作为重要的分子信号或作为防御细胞毒素在生物从古细菌到哺乳动物。真核NOS异构体是大型模块酶；信号酶产生一氧化氮是由钙通过生物传感器钙调素调节NADPH到加氧酶活性位点的电子通量来控制的。控制一氧化氮的产生是许多生理反馈系统的核心。FMN结合域在NADPH脱氢酶和加氧酶催化中心之间穿梭时传递电子。电子从NADPH结合位点进入并在加氧酶位点提供。实验将研究电子进入加氧酶位点状态的形成。电子穿梭的机理将被详细地描述。该项目将重点研究Ca+2/CaM控制的信号发生器nNOS和Ca+2不敏感的iNOS，详细描述穿梭机制及其被钙调蛋白激活的情况。实验计划强调了高年级本科生可以接触到的技术。该提案通过提供NOS功能和控制的新范式以及中心概念的新测试来展示智力价值。基本概念包括系绳穿梭模型，输入和输出状态结构的设计，以及新的动力学模型。作为一个RUI项目，该研究项目有一个完整的培训组成部分，包括本科生参与准备和实验工作。每年夏天至少有四名本科生参加，另外还有学生在学年期间参与该项目。他们将获得制备和实验生物化学和分子遗传学方面的高级培训和经验。学生将学习如何设计一个项目，如何计划和执行一系列实验工作，以及如何分析和评估数据。计算高级的学生将学习模拟的基础知识。学生都将有机会参加地区或国家科学会议并展示工作成果。准备演讲很重要，因为它提供了写作、组织和产生有意义的数字的经验。结果，大约20名不同背景的本科生将被介绍给作为一项活生生的智力活动的科学。他们中的一些人将被招募到科学和相关专业作为职业，但他们所有人都将更好地为自己的职业选择做出决定，并作为公民就地方和国家层面的问题做出决定。

Nitric Oxide synthases produce NO as an important molecular signal or as a defensive cytoxin in organisms ranging from archaea to mammals. Eukaryotic NOS isoforms are large modular enzymes; NO production by signaling enzymes is controlled by calcium regulation of electron flux from NADPH to the oxygenase active site via the biosensor calmodulin. Controlled production of NO is central to many physiological feedback systems. The FMN binding domain delivers electrons as it shuttles between the NADPH dehydrogenase and oxygenase catalytic centers in the enzyme. Electrons enter from the NADPH binding site and are donated at the oxygenase site. Experiments will study the formation of the state in which electrons enter the oxygenase site. The mechanism of the electron shuttle will be described in detail. The project will concentrate on nNOS, a Ca+2/CaM controlled signal generator, and iNOS, which is Ca+2 insensitive, to describe the shuttle mechanism and its activation by calmodulin in detail. The experimental plan emphasizes techniques accessible to advanced undergraduates. The proposal demonstrates intellectual merit by providing new paradigms for NOS function and control and novel tests of central concepts. Underlying concepts include the tethered shuttle model, design of input and output state constructs, and new kinetics models.Broader Impacts As a RUI project, the research program has an integral training component including undergraduate student participation in preparative and experimental work. At least four undergraduate students each summer will participate, and additional students work on the project during the academic year. They will get advanced training and experience in preparative and experimental biochemistry and molecular genetics. Students will learn how to design a project, how to plan and carry out a line of experimental work, and how to analyze and evaluate data. Computationally advanced students will learn the basics of simulation. Students will all have the opportunity to attend a regional or national scientific conference and present work. Preparation of presentations is important because it provides experience in writing, organization, and production of meaningful figures. As a result, about twenty undergraduates with diverse backgrounds will be introduced to science as a living intellectual endeavor. Some of them will be recruited into science and allied professions as a career, but all of them will be better equipped to make decisions about their own career choices and, as citizens, about issues on local and national scale.