Minority Supplement for GM111932

描述(申请人提供)：意义。我们的目标是确定控制酶促一氧化氮解毒的基本结构和热力学特征。我们使用了计算设计和生化分析的循环人工一氧化氮双加氧酶(NOD)形成的人工血红素为基础的氧结合蛋白结构域与黄素蛋白还原酶结构域来自自然。使用这样一种强大、简单的蛋白质使其更容易对蛋白质进行小规模和大规模的改变，并积极确定酶功能所需的关键功能。一氧化氮在人类生物学的许多信号传递过程中发挥着核心作用，但由于其高度的化学反应，它也与许多严重的疾病有关，如Lou Gehrig病和缺血性脑损伤。因此，一种优越的一氧化氮双加氧酶有望在未来许多病理疾病的治疗中发挥作用。相反，不受欢迎的NOD活性已经在以血红蛋白为基础的血液替代品中产生了严重的并发症，学习如何在不对氧结合产生不利影响的情况下降低或消除这些疗法中的NOD活性是很重要的。创新。这种催化结构代表了蛋白质设计的下一代，将设计技术从目前专注于具有单一辅因子的简单蛋白质结构域转移到更具挑战性和更复杂的多结构域结构，这些结构更接近于自然界中看到的复杂组装。该项目有能力极大地推动两项重要技术的发展：基于血红蛋白的血液替代品和酶疗法。首先，从该项目中吸取的经验教训有望重振以血红蛋白为基础的血液替代品领域，使天然血红蛋白的重组成为可能，并创造出在仍携带氧气的情况下与一氧化氮反应最小的全新氧气运输蛋白。其次，合成酶具有改变酶治疗领域的潜力，因为设计的酶比它们的天然酶具有许多优势，最重要的是能够利用非天然辅因子。 更好地优化了目标活性，并大大提高了它们的稳定性，超过天然蛋白(53)。因此，这个项目代表了酶疗法的一个新方向，我们的设计路径是一种使能技术，我们和其他人将在未来的酶疗法的创造中使用它。明确的目标。这项工作将使我们能够回答关于该酶的一些重要问题：目的1.血红素还原潜力在一氧化氮双加氧酶反应中起什么作用？目的2.电子转移动力学和热力学在这个反应中有多重要？目的3.蛋白质动力学和结构是如何控制NOD功能的？

DESCRIPTION (provided by applicant): Significance. We aim to determine the essential structural and thermodynamic features which govern enzymatic nitric oxide detoxification. We use a cycle of computational design and biochemical analysis of an artificial nitric oxide dioxygenase (NOD) formed by combining an artificial heme-based oxygen binding protein domain with a flavoprotein reductase domain derived from nature. Use of such a robust, simple protein makes it significantly easier to make both small- and large-scale changes to the protein and positively identify critical features necessary for enzyme function. Nitric oxide plays a central role in many signaling process in human biology, yet due to its degree of chemical reactivity it has also been implicated in a surprising number of serious disorders such as Lou Gehrig's Disease and ischemic brain injury. A superior nitric oxide dioxygenase thus promises to be useful in future treatments of many pathological conditions. Conversely, unwanted NOD activity has produced severe complications in hemoglobin-based blood substitutes, and it is important to learn how to reduce or eliminate NOD activity in these therapeutics without adversely affecting oxygen binding. Innovation. This catalytic construct represents the next generation in protein design, moving design technology from the current focus on simple protein domains with single cofactors to significantly more challenging and sophisticated multidomain structures that more closely resemble the complex assemblies seen in nature. This project has the capacity to dramatically advance two important technologies: hemoglobin-based blood substitutes and enzyme therapeutics. First, lessons learned in this project promise to revitalize the field of hemoglobin-based blood substitutes, enabling both the reengineering of native hemoglobins and the creation of entirely new oxygen transport proteins minimally reactive with nitric oxide while still carrying oxygen. Second, a synthetic enzyme has the potential to transform the field of enzyme therapy because of the many advantages designed enzymes have over their natural counterparts, most importantly the ability to utilize non-natural cofactors better optimized for the target activity and their greatly increased stability over natural protein (53). This project thus represents a new direction in enzyme therapy, and our design pathway is an enabling technology which will be used by us and others in the creation of future enzyme therapeutics. Specific Aims. This work will allow us to answer some important questions about this enzyme: Aim 1. What role does the heme reduction potential play in the nitric oxide dioxygenase reaction? Aim 2. How important are electron transfer dynamics and thermodynamics in this reaction? Aim 3. How do protein dynamics and structure govern NOD function?