Collaborative Research: Mapping and Tracking Conformational Control of Nitric Oxide Synthase Activation

合作研究：绘制和跟踪一氧化氮合酶激活的构象控制

• 批准号: 1708829
• 负责人: Brian Smith
• 金额: $ 30.06万
• 依托单位: Medical College of Wisconsin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1708829&HistoricalAwards=false
• 关键词: Collaborative; Research; Mapping; Tracking; Conformational

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Carey Johnson from the University of Kansas, Dr. Brian Smith from Medical College of Wisconsin, and Dr. David Arnett from Northwestern College. The project investigates shapes (conformations) and shape changes of the enzyme nitric oxide synthase (NOS). The importance of conformational changes in the function of complex enzymes like NOS has become increasingly recognized. NOS activity requires a sequence of electron transfers between different domains of the enzyme. The hypothesis is that these events are controlled by conformational dynamics. In this project, the conformations and conformational changes of NOS are tracked by time-resolved and single-molecule fluorescence techniques. Mass spectrometry shows how domain interactions are regulated by binding to NOS of a calcium signaling protein, calmodulin. The results may show how multi-domain enzymes function through conformational changes. The project provides training in advanced fluorescence and mass spectrometric techniques for graduate students at Kansas University and the Medical College of Wisconsin. Undergraduate students from all participating institutions are trained in interdisciplinary science research. The project also offers training for members of the UKanTeach program which trains science majors as high school science teachers.This project exploits the spatial and temporal resolution inherent in time-resolved and single-molecule fluorescence to identify and track the conformational states of nitric oxide synthase (NOS). NOS is a homodimeric enzyme that catalyzes formation of nitric oxide by shuttling electrons from modules in one monomer to the heme in the oxygenase domain of the other. Electron transfer in both domains depends on calmodulin binding. Because electron transfer only occurs when the electron donor and electron acceptor are in close proximity, conformational changes must occur to bring different donor and acceptor couples together sequentially. The time scales of interchange among conformational states are not known, nor is it clear how the observed conformational changes correlate with the catalytic cycle of the enzyme. The methods of the project correlate conformations and dynamics detected by fluorescence with knowledge of inter-domain interactions at the peptide level. These inter-domain interactions are observed from hydrogen-deuterium exchange (HDX) mass spectrometry. To identify and assign conformational states observed, selected site-directed mutants that disrupt specific subdomain interactions are probed by both fluorescence and mass spectrometry. The project may lead to a detailed model of NOS function that describes the conformational sequences and rates of conformational changes. The project may also demonstrate how the conformational sequences are related to enzyme regulation. The project provides training in advanced fluorescence and mass spectrometric techniques for graduate students at Kansas University and the Medical College of Wisconsin. Undergraduate students from all participating institutions are trained in interdisciplinary science research. The project also offers training for members of the UKanTeach program which trains science majors as high school science teachers.

通过该奖项，化学过程的化学过程计划是为堪萨斯大学的Carey Johnson博士，威斯康星州医学院的Brian Smith博士和西北学院的David Arnett博士提供资金。该项目研究了形状（构象）和形状的一氧化氮合酶（NOS）的变化。像NOS这样的复杂酶功能上构象变化的重要性已越来越被认可。 NOS活性需要在酶的不同域之间进行一系列电子传输。假设是这些事件是由构象动力学控制的。在这个项目中，通过时间分辨和单分子荧光技术跟踪NOS的构象和构象变化。质谱法显示了如何通过与钙信号蛋白钙调蛋白的NOS结合来调节结构域的相互作用。结果可能显示多域酶如何通过构象变化的功能。该项目为堪萨斯大学和威斯康星州医学院的研究生提供了高级荧光和质谱技术的培训。来自所有参与机构的本科生接受了跨学科科学研究的培训。该项目还为Ukanteach计划的成员提供培训，该计划培训科学专业的高中科学教师。该项目利用了时间分辨和单分子荧光固有的空间和时间分辨率，以识别和跟踪一氮氧化物合酶（NOS）的构象状态。 NOS是一种同型二聚体酶，通过将电子从一个单体中的模块从一个单体中的模块中穿梭到另一个单体的血红素，从而催化一氧化氮的形成。两个结构域中的电子转移取决于钙调蛋白的结合。由于电子传递仅在电子供体和电子受体紧密接近时发生，因此必须发生构象变化，以使不同的供体和受体夫妇顺序依次聚集在一起。构象状态之间互换的时间尺度尚不清楚，也不清楚观察到的构象变化与酶的催化循环如何相关。该项目的方法将荧光检测到的构象和动力学与肽水平上域间相互作用的知识相关联。这些域间相互作用是从氢 - 偏见交换（HDX）质谱法观察到的。为了识别和分配观察到的构象状态，通过荧光和质谱探测了破坏特定亚域相互作用的选定位置定向突变体。该项目可能会导致NOS功能的详细模型，该模型描述了构象序列和构象变化的速率。该项目还可以证明构象序列与酶调节的关系。 该项目为堪萨斯大学和威斯康星州医学院的研究生提供了高级荧光和质谱技术的培训。来自所有参与机构的本科生接受了跨学科科学研究的培训。该项目还为Ukanteach计划的成员提供培训，该计划培训科学专业的高中科学教师。

项目成果

ICEKAT: an interactive online tool for calculating initial rates from continuous enzyme kinetic traces

• DOI: 10.1186/s12859-020-3513-y
• 发表时间: 2020-05-14
• 期刊: BMC BIOINFORMATICS
• 影响因子: 3
• 作者: Olp, Michael D.;Kalous, Kelsey S.;Smith, Brian C.
• 通讯作者: Smith, Brian C.

Conformations and Single-Molecule Dynamics of Nitric Oxide Synthase

一氧化氮合酶的构象和单分子动力学

• DOI: 10.1016/j.bpj.2017.11.3690
• 发表时间: 2018
• 期刊: Biophysical Journal
• 影响因子: 3.4
• 作者: Johnson, Carey K.;Arnett, David C.;Smith, Brian C.
• 通讯作者: Smith, Brian C.

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation

• DOI: 10.1016/j.jmb.2018.02.003
• 发表时间: 2018-03-30
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Hanson, Quinlin M.;Carley, Jeffrey R.;Underbakke, Eric S.
• 通讯作者: Underbakke, Eric S.