He Yufan, Haque Mohammad Mahfuzul, Stuehr Dennis J, Lu H Peter
Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403;
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195.
Proc Natl Acad Sci U S A. 2015 Sep 22;112(38):11835-40. doi: 10.1073/pnas.1508829112. Epub 2015 Aug 26.
Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.
调节一氧化氮合酶（NOS）的机制在生物学和医学领域备受关注。尽管NOS催化依赖于结构域运动，并通过钙调蛋白结合而被激活，但其中的关系尚不清楚。我们使用单分子荧光共振能量转移（FRET）光谱来阐明FRET染料标记的神经元NOS还原酶结构域中两个电子转移结构域的构象状态分布以及相关的构象波动动力学，并了解钙调蛋白如何影响动力学以调节催化作用。我们发现钙调蛋白通过多种方式改变NOS的构象行为：它改变了NOS结构域之间的距离分布，缩短了各个构象状态的寿命，并通过极大地缩小构象状态和波动速率的分布来灌输构象规则性。这些信息只有通过单分子光谱测量才能获得，并揭示了钙调蛋白如何通过塑造NOS的物理和时间构象行为来促进催化作用。
