Conformations and Single-Molecule Dynamics of Nitric Oxide Synthase
Conformations and Single-Molecule Dynamics of Nitric Oxide Synthase
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一氧化氮合酶的构象和单分子动力学
DOI：
10.1016/j.bpj.2017.11.3690
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发表时间：
2018
影响因子：
3.4
通讯作者：
Smith, Brian C.
中科院分区：
文献类型：
--
作者：
Johnson, Carey K.;Arnett, David C.;Smith, Brian C.
一氧化氮合酶的 3392-Pos Board B600 构象和单分子动力学 Carey K. Johnson1、David C. Arnett2、Brian C. Smith3。 1美国堪萨斯州劳伦斯市堪萨斯大学化学系，2美国爱荷华州奥兰治城西北学院化学系，3美国威斯康星州密尔沃基威斯康星医学院生物化学系。一氧化氮合酶 (NOS) 通过电子依次从 FAD 转移到酶还原酶结构域中的 FMN，然后从 FMN 转移到同二聚复合物相对成员的加氧酶结构域中的血红素来发挥作用。有效的电子转移由钙信号蛋白钙调蛋白 (CaM) 激活，并且需要电子转移供体和受体靠近。因此，电子转移的顺序需要酶的多种构象状态，这表明酶的活性是构象门控的。我们通过时间分辨检测来自 CaM 的荧光团的荧光，检测到 NOS 多种构象状态的存在。荧光通过酶的血红素基团的 FRET 猝灭，猝灭的程度取决于酶的构象状态。单分子强度轨迹揭示了毫秒到秒时间尺度上动态的多种荧光状态。分析表明连续构象互换，寿命最长的状态被高度猝灭，这与 CaM 与血红素基团非常接近的构象一致。构象动力学分析正在进行中。
3392-Pos Board B600 Conformations and Single-Molecule Dynamics of Nitric Oxide Synthase Carey K. Johnson1, David C. Arnett2, Brian C. Smith3. 1Chemistry, University of Kansas, Lawrence, KS, USA, 2Chemistry, Northwestern College, Orange City, IA, USA, 3Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA. Nitric oxide synthase (NOS) functions by transfer of electrons sequentially from FAD to FMN in the reductase domain of the enzyme and then from FMN to the heme in the oxygenase domain of the opposing member of a homodimeric complex. Efficient electron transfer is activated by the calcium signaling protein calmodulin (CaM) and requires close proximity of electron-transfer donors and acceptors. The sequence of electron transfers therefore necessitates multiple conformational states of the enzyme, suggesting that the activity of the enzyme is conformationally gated. We have detected the presence of multiple conformational states of NOS by time-resolved detection of fluorescence from a fluorophore attached to CaM. Fluorescence is quenched by FRET to the heme groups of the enzyme, and the extent of quenching depends on the conformational state of the enzyme. Single-molecule intensity trajectories reveal multiple fluorescence states with dynamics on the millisecond to second time scales. Analysis suggests sequential conformational interchange, with the longest-lived state being highly quenched, consistent with a conformation in which CaM is in close proximity with the heme groups. Analysis of conformational dynamics is underway.