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

通过这个奖项，化学学部生命过程化学项目资助了堪萨斯大学的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。该项目研究了一氧化氮合酶（NOS）的形状（构象）和形状变化。像NOS这样的复杂酶的构象变化在其功能中的重要性已被越来越多地认识到。NOS活性需要在酶的不同结构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。在本项目中，利用时间分辨和单分子荧光技术跟踪NOS的构象和构象变化。质谱分析显示结构域相互作用是如何通过与钙信号蛋白钙调蛋白的NOS结合来调节的。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辛医学院的研究生提供先进的荧光和质谱技术培训。所有参与院校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，该项目将科学专业的学生培养成高中科学教师。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。NOS是一种同二聚体酶，通过将电子从一个单体的模组传递到另一个单体的加氧酶结构域的血红素中来催化一氧化氮的形成。两个区域的电子转移依赖于钙调素结合。由于电子转移只发生在电子给体和电子受体靠近时，因此必须发生构象变化才能使不同的电子给体和电子受体依次结合在一起。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肽水平上的域间相互作用的知识联系起来。这些区域间的相互作用是由氢-氘交换（HDX）质谱法观察到的。为了识别和分配观察到的构象状态，选择了破坏特定亚结构域相互作用的位点定向突变体，通过荧光和质谱法进行了探测。该项目可能导致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如何与酶调节相关。该项目为堪萨斯大学和威斯康辛医学院的研究生提供先进的荧光和质谱技术培训。所有参与院校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，该项目将科学专业的学生培养成高中科学教师。

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.