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

• 批准号: 10621327
• 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
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10621327
• 关键词: Alzheimer' 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; Mass Spectrum Analysis; Measurement; Methods; Molecular; Molecular Conformation; Motion; NOS3 gene; Neurons; Nitric Oxide; Nitric Oxide Synthase; Output; Phosphorylation; Phosphoserine; Physiologic pulse; Post-Translational Regulation; Production; Property; Protein Conformation; Protein Dynamics; Protein Isoforms; Proteins; Regulation; Research; Shapes; Site; Spectrum Analysis; Stimulus; Stroke; Structure; System; Techniques; Testing; Work; computational basis; effective therapy; experimental study; flexibility; improved; in vivo; innovation; milligram; novel therapeutic intervention; novel therapeutics; programs; rate of change; response; single-molecule FRET; statistics; synergism; two-dimensional

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 神经元型和内皮型一氧化氮合酶(nNOS和eNOS)产生NO作为对 钙调素(CaM)结合，在人类健康和疾病中具有广泛的功能。翻译后调节 通过磷酸化进一步调节体内一氧化氮合酶对刺激的反应。这些大口味的标志- 血红素蛋白包括具有灵活连接子的多域结构，允许动态、受调节的 域间电子转移(IET)。NO合成需要较大的构象变化，其中FMN 结构域在一氧化氮合酶接受电子的“输入状态”和提供电子的“输出状态”之间穿梭传递 跨域的电子。这些大尺度运动是由构象能量景观塑造的，即， 自由能与蛋白质构象的关系。此外，局部构象调整可能 继续处于停靠状态。尽管进行了广泛的研究，但这些变化背后的动力 IET跨NOS结构域所需的构象变化尚不清楚。回答问题的障碍 这个中心问题是缺乏一个统一的理论/计算方法来解释实验 结果定量分析。解决这一棘手的研究问题需要介观的收敛 对溶液中一氧化氮合酶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一起进行多管齐下的努力是创新的，因为它极大地扩展了 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，并将它们应用于计算的NOS 以一致的方式进行整合行为范例。我们的综合计划借鉴了独特的 协作团队的综合专业知识。重要的是，我们已经迈出了关键的第一步 我们的实验和计算方法是协同的。 为了确定能量格局和由此产生的NOS构象属性，我们将首先计算 构象统计和动力学，并将其与合适的实验协同使用来研究远程 在不同的一氧化氮合酶蛋白中系留结构域运动。此外，我们还将研究局部构象 停靠状态下的调整。然后，我们将应用我们的综合方法来研究 通过功能上重要的磷酸化的构象景观。总而言之，这些结果将提供 对作为一氧化氮合酶机制核心部分的蛋白质动力学有一个全面的定量理解。这个 拟议的研究具有重要意义，因为它将回答关于一氧化氮合酶亚型的长期存在的基本问题 通过定义管理强制性环境的构象方面(统计、动态和能源格局) 一氧化氮合酶中的电子转移步骤。这项工作将积极影响我们对其他生物分子的理解，如 确定结构-动力学-功能关系是当前生化研究的核心。

项目成果

Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.

• DOI: 10.1021/acs.jpcb.2c04091
• 发表时间: 2022-09-15
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY B
• 影响因子: 3.3
• 作者: Tumbic, Goran W.;Li, Jinghui;Jiang, Ting;Hossan, Md Yeathad;Feng, Changjian;Thielges, Megan C.
• 通讯作者: Thielges, Megan C.

An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase.

• DOI: 10.1007/s00775-020-01824-w
• 发表时间: 2020-12
• 期刊: Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
• 作者: Zheng H;Li J;Feng C
• 通讯作者: Feng C

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases.

• DOI: 10.1016/j.jinorgbio.2020.111298
• 发表时间: 2021-01
• 期刊: Journal of inorganic biochemistry
• 影响因子: 3.9
• 作者: Zheng H;Weaver JM;Feng C
• 通讯作者: Feng C

Probing Protein Dynamics in Neuronal Nitric Oxide Synthase by Quantitative Cross-Linking Mass Spectrometry.

• DOI: 10.1021/acs.biochem.3c00245
• 发表时间: 2023-08-01
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Jiang, Ting;Wan, Guanghua;Zhang, Haikun;Gyawali, Yadav Prasad;Underbakke, Eric S.;Feng, Changjian
• 通讯作者: Feng, Changjian