Imaging of protein dimerization using GFP mutants

至于担负细胞增殖、分化的信号换能器的增强机制，也有许多问题尚不清楚。有一些直接的结果，处理了分子内构象变化和分子间结合的实时刺激以及由于修饰（如磷酸化）而引起的增强。认为信号换能器的二聚化作为增强机制之一发挥了至关重要的作用。FRET是用来可视化二聚化可见性。将GFP突变体与目标肽融合以进行荧光标记。这两年的研究成果如下：(1)建立了一种利用荧光蛋白与FRETIn测定蛋白相互作用的方法，为了验证荧光蛋白标记方法的有效性，对DMA gyraseB调光剂之间的相互作用进行了评估。在溶液中观察到更多的GyrB-CFP和GyrB-YFP在二聚化作用下形成一对。(2)利用GFP及其突变体进行单分子鉴定——创造了新的GFP突变体（未销售），其荧光光谱波长介于CFP和GFP之间，并且/或者可以在单分子水平上成像。此外，激光显微镜的发展可以在单分子水平上直接可见，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，并成功地在单分子水平上看到了GFP和YFP的二聚化。(3)观察活细胞中YFP-Ras和GFP-Raf1动态的时空相关性，包括FRET成像。在EGF刺激后，Raf1直接与质膜上的Ras相互作用。如上所述，证明了GFP突变体在单分子水平上可以作为二聚化的分子标记。如果二聚化和增强之间的关系可以在活细胞中显示出来，我目前研究了它的可能性，并想把它与细胞功能成像的应用联系起来。少

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