2001 Fiscal Year Final Research Report Summary
Development of time-resolved spectroscopic observation systems for the fast dynamics of protein folding
| Project/Area Number |
12558081
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biophysics
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
TAKAHASHI Satoshi kyoto University, Graduate School of Engineering, Instructor, 工学研究科, 助手 (30283641)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIMORI Koichiro kyoto University, Graduate School of Engineering, Assistant Professor, 工学研究科, 助教授 (20192487)
HARADA Yoshiyuki Shimadzu Corporation, 分析機器事業部, 副主任研究員
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| Project Period (FY) |
2000 – 2001
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| Keywords | protein folding / time-resoloved observation / rapid mixing device / CD spectrometer / cytochrome c / poly-L-glutamic acid / apomyoglobin |
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| Research Abstract |
We developed the following original instruments to observe the early dynamics of protein folding. 1) The rapid mixing device that can initiate protein folding within 50 μs. The previous mixing devices has the mixing time of 300〜400 μs. Thus, we achieved to reduce the mixing times to nearly an order shorter. 2) We constructed to the observation system for the CD spectroscopy based on the prism polychrometer and a CCD detector. We resolved several problems associated with the new ideas of spectroscopic observation system, such as the effect of optical retardation. The developed system enabled us to observe the circular dichroism spectra with a high S/N ratio based on the multichannel detection. 3) We tried to combine the developped flow cell system and the spectroscopic observation system ; however, faild to observe reliable data for the repid-mixing cell. We are currently working to reduce the optical retardations of the cells.
Using the developed flow cell systems, we obtained the follo
… More
wing results that were already reported in scientific journals. 1) We observed time-resolved small-angle X-ray scattering profiles for the protein folding dynamics of cytochrome c. The developed cell enabled us to obtain information on the early submillisecond phase of cytohrome c folding that were inaccessible by the conventional devices. We conclude that the protein compaction and the secondary structure formations proceed simultaneously in the folding process of cytochrome c. 2) We observed the helix formation processes of polyglutamic acid using the developed rapid-mixing cell. We demonstrated that a short helix formation precedes the longer helix formation process in the PGA helix formation. This is a new dynamical event observed in the helix formation of polypeptides. 3) The helix-formation and the collapse process of apomyoglobin were observed using the developed rapid mixing systems in the submillisecond time domain. We clarified that the apomyoglobin folding proceed as a stepeise process that is similar to the observation for cytochrome c. Less
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