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.

该奖项将资助堪萨斯大学的Carey Johnson博士、威斯康星医学院的Brian Smith博士和西北学院的David Arnett博士。该项目研究一氧化氮合酶(NOS)的形状(构象)和形状变化。像一氧化氮合酶这样的复杂酶功能的构象变化的重要性已经被越来越多地认识到。一氧化氮合酶的活性需要在酶的不同区域之间进行一系列的电子转移。假设这些事件是由构象动力学控制的。本项目利用时间分辨和单分子荧光技术跟踪一氧化氮合酶的构象和构象变化。质谱仪显示了钙信号蛋白钙调蛋白与一氧化氮合酶结合是如何调节结构域相互作用的。这些结果可能表明多结构域酶是如何通过构象变化发挥作用的。该项目为堪萨斯大学和威斯康星医学院的研究生提供先进的荧光和质谱学技术培训。所有参与机构的本科生都接受跨学科科学研究方面的培训。该项目还为UKanTeach项目的成员提供培训，该项目将理科专业的学生培训为高中理科教师。该项目利用时间分辨和单分子荧光固有的空间和时间分辨率来识别和跟踪一氧化氮合酶(NOS)的构象状态。一氧化氮合酶是一种同源二聚体酶，通过将电子从一个单体的模块传递到另一个单体加氧酶结构域的血红素，催化一氧化氮的形成。这两个结构域的电子传递都依赖于钙调蛋白结合。由于电子转移只有当电子给体和电子受体靠近时才发生，所以必须发生构象变化才能使不同的给体和受体对顺序地结合在一起。构象状态之间交换的时间尺度尚不清楚，也不清楚所观察到的构象变化如何与酶的催化循环相关。该项目的方法将荧光检测到的构象和动力学与多肽水平上的结构域间相互作用的知识联系起来。这些区域间的相互作用是通过氢-氚交换(HDX)质谱学观察到的。为了识别和分配观察到的构象状态，通过荧光和质谱学来探测破坏特定亚域相互作用的选定的定点突变体。该项目可能导致一个详细的一氧化氮合酶功能模型，描述构象序列和构象变化的速率。该项目还可能展示构象序列如何与酶调节有关。该项目为堪萨斯大学和威斯康星医学院的研究生提供先进的荧光和质谱学技术培训。所有参与机构的本科生都接受跨学科科学研究方面的培训。该项目还为UKanTeach计划的成员提供培训，该计划将理科专业的学生培训为高中理科教师。

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.

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.

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.