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 神经元和内皮一氧化氮（NO）合酶（nNOS和eNOS）酶使NO响应于 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NOS在体内对刺激的反应。这些大口味的标志- 血红素蛋白包括具有柔性接头的多结构域结构，允许动态的、受调节的 畴间电子转移（IET）。NO合成需要大的构象变化，其中FMN 域之间穿梭NOS的电子接受“输入状态”和电子捐赠“输出状态”，以提供 电子穿过域。这些大规模的运动是由构象能量景观塑造的，即， 自由能对蛋白质构象的依赖性。此外，局部构象调整可能 继续处于对接状态。尽管进行了广泛的研究，但这些研究背后的动力学 跨NOS结构域的IET所需的构象变化仍不清楚。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 定量的结果。解决这个令人烦恼的研究问题需要一个介观的收敛 计算分析和动手实验，是敏感的NOS蛋白质动力学的解决方案。 将这些最新的实验方法结合在一起，多管齐下，这是一种创新， 实验测量的整体范围，并提供了一个更好的基础计算。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 以一致的方式进行构象行为范式。我们的综合方案借鉴了独特的 合作团队的专业知识。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的协同作用。 为了确定能量分布和由此产生的NOS构象性质，我们将首先计算能量分布。 构象统计学和动力学，并将其与合适的实验协同使用，以研究长程 各种NOS蛋白质中的栓系结构域运动。此外，我们将研究局部构象 对接状态下的调整。然后，我们将应用我们的综合方法来研究重塑的 构象景观的功能重要的磷酸化。综合起来，这些结果将提供 蛋白质动力学作为NOS机制的核心部分的全面定量理解。的 一项拟议中的研究意义重大，因为它将回答有关NOS亚型的长期存在的基本问题 通过定义支配强制性的构象方面（统计、动力学和能量景观） 电子转移步骤NOS。这项工作将积极影响我们对其他生物分子的理解， 定义结构-动力学-功能关系是当前生物化学研究的核心。

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