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Imaging of protein dimerization using GFP mutants

使用 GFP 突变体对蛋白质二聚化进行成像

基本信息

批准号：
12680656
负责人：
IWANE Atsuko
金额：
$ 0.64万
依托单位：
Osaka University Graduate School of medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

As for the potentiation mechanism of the signal transducer which shoulders the proliferation, differentiation of the cell, there are many points which are still unclear, too. There are a few direct results, which cope with the stimulation of the realtime for intramolecular conformational change and intermolecular association and the potentiation due to the modification such as a phosphorylation as well. It is said that the role that dimerization of the signal transducer is critical is fulfilled as one of the potentiation mechanisms. FRET is used to be visualized the dimerization visibility. GFP mutants was fused with the target peptide for the fluorescent labeling.The achievements of this study for these two years are listed below.(1) Development of a method for measuring protein interaction using fluorescent proteins and FRETIn order to validate the method for fluorescent protein labeling, an interaction between DMA gyraseB dimmer was assessed. Using coumermysin as a linker molecule, … More a pair of GyrB-CFP and GyrB-YFP was formed FRET caused by dimerization was observed in solution.(2) Single-molecular identification by GFP and its mutantsI created new GFP mutants (isn't merchandised), having fluorescent spectrum wavelength between CFP and GFP and/or can be imaging at single molecular level Furthermore, the development of the laser microscope which can be visualizeble in directly at single molecular level to estimate the order of the number of molecules. I showed the fluorescent characters of these GFP mutants and succeeded in making it visibility the dimerization of CGFP and YFP at single molecular level.(3) Spatio-temporal correlation between the dynamics of YFP-Ras and GFP-Raf1 in living cells including FRET imaging was observed. After the EGF stimulation, Raf1 directly interacted with Ras at the plasma membrane. It was proved that GFP mutant was useful as molecular tagging of dimerization at single molecular level as mentioned above. If the relationship between the dimerization and the potentiation can be showed in the living cell, Ifm examined its possibility at present and want to connect it to the applicatio to the imaging of the cell function. Less

对于肩负着细胞增殖、分化的信号转导子的增强机制，也有许多不清楚的地方。有几个直接的结果，科普刺激的实时分子内构象变化和分子间的协会和增强由于修饰，如磷酸化以及。据说，信号转导子的二聚化是关键的作用作为增强机制之一而实现。FRET用于可视化二聚化可见性。将GFP突变体与靶肽融合，进行荧光标记。(1)为了验证荧光蛋白标记的方法，评估了DMA gyrase B二聚体之间的相互作用。使用香豆素作为连接分子， ...更多信息形成一对GyrB-CFP和GyrB-YFP，在溶液中观察到由二聚化引起的FRET。(2)通过GFP及其突变体的单分子鉴定我创造了新的GFP突变体（未商品化），具有CFP和GFP之间的荧光光谱波长和/或可以在单分子水平上成像。此外，激光显微镜的发展可以直接在单分子水平上可视化，以估计分子数量的顺序。我展示了这些GFP突变体的荧光特性，并成功地使其在单分子水平上可见CGFP和YFP的二聚化。(3)观察到包括FRET成像在内的活细胞中YFP-Ras和GFP-Raf 1动力学之间的时空相关性。EGF刺激后，Raf 1直接与Ras在质膜上相互作用。证明了GFP突变体可用于上述单分子水平上的二聚化的分子标记。如果二聚化和增强之间的关系可以在活细胞中显示，Ifm目前正在研究其可能性，并希望将其应用于细胞功能成像。少