2001 Fiscal Year Final Research Report Summary
Imaging of protein dimerization using GFP mutants
| Project/Area Number |
12680656
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University Graduate School of medicine |
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Principal Investigator |
IWANE Atsuko Graduate School of Medicine, Osaka University, Assistant Professor, 医学系研究科, 助手 (30252638)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Green Fluorescent Protein / Single Molecule Imaging / Dimerization / Fluorescent Energy Transfer |
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| Research Abstract |
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 FRET
In order to validate the method for fluorescent protein labeling, an interaction between DMA gyraseB dimmer was assessed. Using coumermysin as a linker molecule,
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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 mutants
I 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
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Research Products
(12 results)
