| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The observation of the molecular motion and function at single molecule level allows us to know the dynamics of individual molecules, whereas the conventional methods give average values of the system where protein molecules are placed. However, chemically labeling proteins with fluorescent dyes has the potential to damage protein microstructure, which could in turn alter protein function. To minimize this problem, we have considered to label proteins with biofluorescent molecules, GFP (green fluorescent protein).
The achievements of this study for these two years are listed below.
(1) Development of in vitro protein synthesis system for preparing labeled proteins
The gene expression system using conventional methods were found inadequate for the preparation of recombinant skeletal muscle myosin subfragment-1 (S1) in the biologically active form. In vitro protein synthesis was able to produce the fusion protein of S1 and GFP with biological functions. Individual ATP turnover by S1-GFP all
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ow us to assay the function of a protein by visualizing association-(hydrolysis)-dissociation of the fluorescent ATP analogue, Cy3-ATP.
(2)Single-molecular identification by fluorescent proteins other than GFP
Using other fluorescent molecules (green fluorescent protein mutant EYFP, sea urchin derived red fluorescent protein DsRed), individual single molecules were clearly visualized under low background total internal reflection fluorescence microscopy.
(3) Development of a method for measuring molecular interaction by FRET
In order to validate the method for fluorescent protein labeling, an interaction between DNA gyrase B dimer was assessed. Using coumermysin as a linker molecule, a pair of GyrB-CFP and GyrB-YFP or a pair of GyrB-GFP and GyrB-DsRed was formed. FRET was observed in each pair in solution without any interference as clear as do the molecules labeled by the conventional methods.
In conclusion, proteins labeled with GFP or its mutants are appropriate for observation at single molecular level. This method is expected to widely apply to research the protein dynamics into the cell at single-molecular level. Less
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