2000 Fiscal Year Final Research Report Summary
Time-Resolved Vibrational Spectroscopic Study of Higher Order Structural Changes of Protein Induced by Nanosecond Temperature Jump.
| Project/Area Number |
10480187
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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 | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Okazaki National Research Institutes |
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Principal Investigator |
KITAGAWA Teizo Okazaki National Research Institutes, 統合バイオサイエンスセンター, 教授 (40029955)
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| Co-Investigator(Kenkyū-buntansha) |
OGURA Takashi The University of Tokyo, 大学院・総合文化研究科, 助教授 (70183770)
MIZUTANI Yashisa Okazaki National research Institutes, 分子科学研究所, 助手 (60270469)
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| Project Period (FY) |
1998 – 2000
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| Keywords | Temperature jump / Time-resolved Raman / Ribonuclease / Thermal unfolding of protein / nanosecond T-jump / time-resolved vibrational spectra / thermal denaturation of protein |
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| Research Abstract |
A nanosecond temperature jump (T-jump) apparatus was constructed and combined with time-resolved Raman measurements to investigate thermal unfolding of a protein for the first time. The 1.56 μm-heat pulse with 9 ns width, 10 Hz was obtained through the two-step stimulated Raman scattering in D_2 gas involving seeding and amplification. To achieve uniform temperature rise, the counter-propagation geometry was adopted. The temperature rise was determined by anti-Stokes to Stokes intensity ratios of the 317 and 897 cm^<-1> bands of MoO^<2->_4 aqueous solution. The T-jump as large as 9℃ in 10 ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532 nm. In the initial 200 ns after T-jump, the C-S stretch of Met residues exhibited 10% change of that expected from the steady state spectra and another 10% in 5 ms. The Raman intensity of S0^<2->_4 ions around 980 cm^<-1> increased at 100 μs delay and was shifted to a lower frequency than the steady state frequency of the elevated temperature, presumably due to partial release from the active site. The S-S stretches and tyrosine doublets displayed little change within 5 ms. Thus, the conformation change in the initial step of unfolding is not always concerted.
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