| Research Abstract |
Development of a high sensitive and high time-resolved fluoresce measurement system
We developed a new fluorescence measurement system with high-time resolution and high sensitivity. This system allowed us to measure the fluorescence of a sample even contained a photolabile molecule, rhodopsin, with millisecond time resolution. We measured the transient fluorescence change of octopus rhodopsin, labeled by fluorescence dye specifically at Cys345 of the C terminal, upon light activation. It was found that the fluorescence of the dye changed with the time constant of about 1 msec. On the other hand, the absorptional change, reflecting the micro environment around the retinal chromophore, was completed within 100 msec. These results indicated that the conformational change around C-terminal took place after the conformational change around a retinal. It was suggested that the C-terminal conformational change may be involved in the interaction with G-protein.
The real-time measurement of the activation of G-protein by using fluorescence labeled GTP analogue
So far, it has been known that the intrinsic fluorescence of G-protein can monitor its active or inactive state. However, the detection is quite difficult under the circumstance mixed with other proteins like receptor. To circumvent this problem, we tried to detect the selective fluorescence from the only GTP bound to G-protein by using the fluorescent GTP analogue (mant (N-Methyl-3'-O-anthranoyl)-GTP, mant-GTPyS) on the basis of the fluorescence energy transfer principle. Consequently, we succeeded in recording, in real time, the GDP/GTP exchange reaction process upon photo-activation of rhodopsin. Furthermore, this method can also monitor GTP hydrolyzed process, because G-protein has so low affinity for the hydrolyzed mant-GDP that it rapidly release from G-protein, accompanied by the fluorescence decaying.
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