Defining the conformational control of nitric oxide synthases by a multipronged approach

通过多管齐下的方法定义一氧化氮合酶的构象控制

基本信息

批准号：
10404575
负责人：
Changjian Feng
金额：
$ 31.42万
依托单位：
UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10404575
关键词：
Alzheimer&apos s Disease Anabolism Architecture Back Behavior Binding Biochemical Calmodulin Complex Computer Analysis Computer Models Dependence Deuterium Development Disease Docking Electron Spin Resonance Spectroscopy Electron Transport Electrons Enzymes Family Flavin Mononucleotide Free Energy Genetic Code Health Heart Heme Hemeproteins Human Hydrogen Kinetics Knowledge Lead Mass Spectrum Analysis Measurement Methods Molecular Molecular Conformation Motion NOS3 gene Neurons Nitric Oxide Nitric Oxide Synthase Output Oxidants Phosphorylation Phosphoserine Physiologic pulse Post-Translational Regulation Production Property Protein Conformation Protein Dynamics Protein Isoforms Proteins Regulation Research Site Spectrum Analysis Stimulus Stroke Structure System Techniques Testing Work base computational basis effective therapy experimental study flexibility in vivo innovation milligram novel therapeutic intervention novel therapeutics programs rate of change response single-molecule FRET statistics synergism two-dimensional vibration

Alzheimer&apos s Disease Anabolism Architecture Back Behavior Binding Biochemical Calmodulin Complex Computer Analysis Computer Models Dependence Deuterium Development Disease Docking Electron Spin Resonance Spectroscopy Electron Transport Electrons Enzymes Family Flavin Mononucleotide Free Energy Genetic Code Health Heart Heme Hemeproteins Human Hydrogen Kinetics Knowledge Lead Mass Spectrum Analysis Measurement Methods Molecular Molecular Conformation Motion NOS3 gene Neurons Nitric Oxide Nitric Oxide Synthase Output Oxidants Phosphorylation Phosphoserine Physiologic pulse Post-Translational Regulation Production Property Protein Conformation Protein Dynamics Protein Isoforms Proteins Regulation Research Site Spectrum Analysis Stimulus Stroke Structure System Techniques Testing Work base computational basis effective therapy experimental study flexibility in vivo innovation milligram novel therapeutic intervention novel therapeutics programs rate of change response single-molecule FRET statistics synergism two-dimensional vibration

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项目摘要

Project Summary The neuronal & endothelial nitric oxide (NO) synthase (nNOS & eNOS) enzymes make NO in response to calmodulin (CaM) binding, and function broadly in human health and disease. Posttranslational regulation through phosphorylation further regulates NOS in vivo in response to stimuli. Hallmarks of these large flavo- hemoproteins include multi-domain architecture with flexible linkers, allowing for dynamic, regulated interdomain electron transfer (IET). NO synthesis requires a large conformational change, in which the FMN domain shuttles between NOS's electron-accepting “input state” and electron-donating “output state” to deliver electrons across the domains. These large-scale motions are shaped by conformational energy landscape, i.e., the dependence of free energy on protein conformation. Moreover, local conformational adjustment likely continues in the docked state. Despite extensive research efforts, the dynamics underlying these conformational changes required for IET across the NOS domains remain unclear. A roadblock to answering this central question is the lack of a unified theoretical/computational approach to interpret the experimental results quantitatively. Solving this vexing research problem calls for a convergence of mesoscopic computational analysis and hands-on experiments that are sensitive to NOS protein dynamics in solution. Combining these latest experimental methods in a multipronged effort is innovative, as it dramatically expands the overall scope of the experimental measurements and provides a better basis for the computations. This approach will allow us to interpret the diverse experimental results and apply them to the calculated NOS conformational behavior paradigm in a consistent manner. Our integrated program draws on the unique combined expertise of the collaborative team. Importantly, we have made the crucial first step of implementing our experimental and computational approaches synergistically. To determine the energy landscape and the resulting NOS conformational properties, we will first calculate the conformational statistics and dynamics and use it in synergy with the suitable experiments to study long-range tethered domain motions in various NOS proteins. Furthermore, we will investigate local conformational adjustments in the docked state. We will then apply our integrated approach to study remodeling of the conformational landscape by functionally important phosphorylation. Taken together, these results will provide a comprehensive quantitative understanding of protein dynamics as a central part of NOS mechanisms. The proposed research is significant as it will answer long-standing fundamental questions about the NOS isoforms by defining the conformational aspects (statistics, dynamics, and energy landscape) that govern the obligatory electron transfer steps in NOS. This work will positively impact our understanding of other biomolecules as defining structure-dynamics-function relationship lies at the heart of current biochemical research.

项目摘要神经元和内皮一氧化氮（NO）合酶（NNOS和ENOS）酶不适合对钙调蛋白（CAM）结合，并在人类健康和疾病中广泛发挥作用。翻译后调节通过磷酸化，进一步调节了对刺激的体内NOS。这些大的Flavo-的标志血蛋白包括具有柔性接头的多域结构，允许动态，调节域间电子传输（IET）。没有合成需要大的构象变化，其中FMN NOS的电子感受的“输入状态”和持光的“输出状态”之间的域名跨域的电子设备。这些大规模运动是由构象能景观形成的，即自由能对蛋白质构象的依赖性。此外，本地构象调整可能在停靠的状态下继续。尽管进行了广泛的研究工作，但这些动态是这些的动态 IET跨NOS域所需的构象变化尚不清楚。回答的障碍这个中心问题是缺乏解释实验的统一理论/计算方法定量结果。解决这个烦恼的研究问题需要介绍介绍对溶液中NOS蛋白动力学敏感的计算分析和动手实验。将这些最新的实验方法结合在多重工作中是创新的，因为它大幅扩展实验测量的总体范围，为计算提供了更好的基础。这方法将使我们能够解释不同的实验结果并将其应用于计算的NOS 以一致的方式构象行为范式。我们的集成程序借鉴了独特的合作团队的联合专业知识。重要的是，我们是实施的关键第一步我们的实验和计算方法是协同的。为了确定能量格局和所得的NOS构象性能，我们将首先计算构象统计和动力学，并与合适的实验协同使用它来研究远程各种NOS蛋白中的束缚结构域运动。此外，我们将研究当地构象对接状态的调整。然后，我们将采用综合方法来研究重塑通过功能重要的磷酸化构象局势。综上所述，这些结果将提供对蛋白质动力学的全面定量理解是NOS机制的核心部分。拟议的研究很重要，因为它将回答有关NOS同工型的长期基本问题通过定义管理强制性的构象方面（统计，动态和能源格局）电子传输步骤中的NOS。这项工作将对我们对其他生物分子的理解产生积极影响定义结构 - 动力学功能关系是当前生化研究的核心。