Imaging of protein dimerization using GFP mutants

至于担负细胞增殖、分化的信号转导的增强机制，目前也有许多不清楚的地方。有几个直接的结果，涉及分子内构象变化和分子间结合的实时刺激，以及由于磷酸化等修饰而产生的增强作用。据说，信号转导的二聚化是作为增强机制之一来发挥关键作用的。FRET用于可视化二聚化可见性。本研究的主要研究成果如下：(1)建立了一种利用荧光蛋白和Fretin来测量蛋白质相互作用的方法。为了验证荧光蛋白标记方法的有效性，对DMA gyraseB二聚体之间的相互作用进行了评估。使用Coumermysin作为连接物分子，…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)，其荧光光谱波长介于CFP和GFP之间，和/或可以在单分子水平上成像。此外，可以直接在单分子水平上可视化的激光显微镜的研制，以估计分子的数量顺序。我展示了这些GFP突变体的荧光特征，并成功地在单分子水平上显示了CGFP和YFP的二聚化。(3)包括FRET成像在内的YFP-RAS和GFP-Raf1在活细胞中的动态变化具有时空相关性。经EGF刺激后，Raf1与Ras在质膜上直接相互作用。以上结果表明，GFP突变体在单分子水平上可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，IFM目前研究了它的可能性，并希望将其与细胞功能成像的应用联系起来。较少

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