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

有了这个奖项，化学部的生命过程化学项目资助了来自堪萨斯大学的凯里约翰逊博士、来自威斯康星州医学院的布赖恩史密斯博士和来自西北学院的大卫阿内特博士。该项目研究了一氧化氮合酶（NOS）的形状（构象）和形状变化。构象变化在复合酶如NOS的功能中的重要性已越来越被认识到。NOS活性需要酶的不同结构域之间的电子转移序列。假设这些事件受构象动力学控制。本研究利用时间分辨荧光技术和单分子荧光技术对一氧化氮合酶的构象和构象变化进行了跟踪研究。质谱显示了如何通过与钙信号蛋白钙调素的NOS结合来调节结构域相互作用。这些结果可以显示多结构域酶如何通过构象变化发挥功能。该项目为堪萨斯大学和威斯康星州医学院的研究生提供高级荧光和质谱技术培训。所有参与机构的本科生都接受跨学科科学研究的培训。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养为高中理科教师。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。NOS是一种同二聚体酶，其通过将电子从一个单体中的模块穿梭到另一个单体的加氧酶结构域中的血红素来催化一氧化氮的形成。两个结构域中的电子转移依赖于钙调素结合。因为电子转移只发生在电子供体和电子受体非常接近的时候，所以必须发生构象变化才能将不同的供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。该项目的方法将通过荧光检测的构象和动力学与肽水平上的结构域间相互作用的知识相关联。从氢-氘交换（HDX）质谱法观察到这些结构域间相互作用。为了鉴定和分配观察到的构象状态，通过荧光和质谱法探测破坏特定亚结构域相互作用的所选定点突变体。该项目可能会导致一个详细的模型NOS功能，描述的构象序列和构象变化率。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星州医学院的研究生提供高级荧光和质谱技术培训。所有参与机构的本科生都接受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，该计划将科学专业的学生培养为高中科学教师。

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.