Yang Yoosoo, Heo Paul, Kong Byoungjae, Park Jun-Bum, Jung Young-Hun, Shin Jonghyeok, Jeong Cherlhyun, Kweon Dae-Hyuk
Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, South Korea.
Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746, South Korea.
Biochem Biophys Res Commun. 2015 Oct 2;465(4):864-70. doi: 10.1016/j.bbrc.2015.08.111. Epub 2015 Aug 28.
Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.
可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白产生膜融合所需的能量。它们形成一个平行排列的四螺旋束，称为SNARE复合体，其形成从N端开始，朝着膜近端的C端进行。此前，我们已经表明，这种拉链样过程可由几种黄酮类化合物控制，这些黄酮类化合物与SNARE拉链过程中形成的中间结构结合。在这里，我们的目的是测试在内侧小叶混合试验中观察到的荧光共振能量转移信号是否确实代表半融合囊泡。我们表明，双层膜融合时伴随的囊泡大小变化是膜融合进程的一个良好指标。随着膜融合从囊泡对接发展到半融合和完全融合，两个直径相同为D的融合囊泡的流体动力学直径分别呈现为2D + d（d为膜间距离）、2D和2D。膜融合的动态光散射分析表明，杨梅素在半融合状态下阻止膜融合，而飞燕草素和花青素则阻止囊泡对接。这些结果与我们之前在荧光共振能量转移分析中的发现一致。
