| Project/Area Number |
13354010
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
動物生理・代謝
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SHICHIDA Yoshinori Kyoto University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (60127090)
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| Co-Investigator(Kenkyū-buntansha) |
HORIGUCHI Chiyoharu Hamamatsu Photonics KK, Acting group manager, システム(事)・第1設計部, グループ長代理
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥51,870,000 (Direct Cost: ¥39,900,000、Indirect Cost: ¥11,970,000)
Fiscal Year 2003: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2002: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000)
Fiscal Year 2001: ¥39,780,000 (Direct Cost: ¥30,600,000、Indirect Cost: ¥9,180,000)
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| Keywords | rhodopsin / absorption spectroscopy / fluorescence spectroscopy / CCD camera / optical technology / photoreceptor protein / G-protain / GPCR / 工学技術 / 高速分光 / 可視吸収 / 蛍光 / 蛋白質 / 中間体 / 分光学 / イメージインテンシファイア / トランスデューシン / 分光光度計 / 蛋白質間相互作用 |
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| Research Abstract |
Signal transductions in biological systems are initiated by conformational changes of receptor proteins followed by the interaction with, in most case, other proteins. In this research project, we aimed to develop new equipment for monitoring the conformational changes f photoreceptor proteins and their interactions with other proteins under the physiological conditions. We tried to develop a high performance absorption/fluorescence spectrophotometer using a cooled CCD camera as a multi-channel detector. We have succeeded in developing a spectrophotometer that can continuously record the spectra with a time resolution of 10 ms, which is about 10,000 times higher than that of a conventional spectrophotometer that has a single-channel detector. By introducing the monitor beam and excitation laser, optimization of the optics and software, this equipment allowed to measuring the 200 absorption spectra of the visual pigment rhodopsin (quantum yield 〜0.67) with low bleaching (〜3 %) and low baseline drift (〜0.002 OD) during 〜1hr recording time before and after light excitation. The accuracy of the data obtained by the recording condition is comparable to that obtained from conventional spectrophotometers except for the time resolution.
This system allowed us to monitor the interaction process of rhodopsin with G-protein transducin in photoreceptor membranes under the physiologically relevant conditions. In addition, we were able to monitor the binding and the releasing processes of ligand retinal into/from protein as fluorescence changes of the intrinsic tryptophan residues. These results indicate that this spectrophotometer would be a useful tool for furthering our understanding of the molecular mechanisms of biological signal transduction under the physiological conditions.
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