Time-Resolved Vibrational Spectroscopic Study of Higher Order Structural Changes of Protein Induced by Nanosecond Temperature Jump.

纳秒温度跳跃引起的蛋白质高阶结构变化的时间分辨振动光谱研究。

• 批准号: 10480187
• 负责人: KITAGAWA Teizo
• 金额: $ 6.98万
• 依托单位: Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10480187/
• 关键词: Temperature jump; Time-resolved Raman; Ribonuclease; Thermal unfolding of protein; nanosecond T-jump; time-resolved vibrational spectra; thermal denaturation of protein; 熱変性; 蛋白質アンファルディング; 蛋白質高次構造変化; 蛋白質フォルディング

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.

构建了纳秒温度跳跃（T-jump）装置，并将其与时间分辨拉曼测量相结合，首次研究蛋白质的热解折叠。通过D_2气体中种子和放大两步受激拉曼散射，获得了宽度为9 ns、频率为10 Hz的1.56 μm热脉冲。为了实现均匀的温升，采用了反向传播几何结构。通过MoO 2-4 水溶液的317和897cm ^ -1 谱带的反斯托克斯与斯托克斯强度比来确定温度升高。在10 ns内实现了高达9℃的T跳跃。使用 532 nm 激发的时间分辨拉曼光谱监测牛胰腺核糖核酸酶 A 的展开。在 T 跳跃后的最初 200 ns 中，Met 残基的 C-S 拉伸表现出稳态光谱预期变化 10%，并在 5 ms 内又表现出 10%。 980 cm^ -1 附近的S0^<2->_4离子的拉曼强度在100μs延迟时增加，并且移动到比升高的温度的稳态频率更低的频率，大概是由于活性位点的部分释放。 S-S 延伸和酪氨酸双联体在 5 毫秒内几乎没有变化。因此，展开初始步骤中的构象变化并不总是一致的。

项目成果

K.Yamamoto et al.: "Construction of Novel Nanosecond Temperature Jump Apparatuses Applicable to Raman Mesurements and Direct Observation of Transient Temperature"Applied Spectroscopy. 54. 1591-1604 (2000)

K.Yamamoto 等人：“适用于拉曼测量和瞬态温度直接观察的新型纳秒温跃装置的构造”应用光谱学。

K.Yamamoto, Y.Mizutani, and T.Kitagawa: "Constrctuion of novel nanosecond temperature jump apparatuses applicable to Raman measurements and direct observation of transient temperature."Applied Spectroscopy. 54. 1591-1604 (2000)

K.Yamamoto、Y.Mizutani 和 T.Kitakawa：“构建适用于拉曼测量和瞬态温度直接观察的新型纳秒温度跳跃装置。”应用光谱学。

K.Yamamoto et al.: "Nanosecond Temperature Jump and Time-resolved Raman Study of Thermal Unfolding of Ribonuclease A"Biophysical Journal. 79. 485-495 (2000)

K.Yamamoto 等人：“核糖核酸酶 A 热展开的纳秒温度跳跃和时间分辨拉曼研究”生物物理学杂志。

K.Yamamoto, Y.Mizutani, and T.Kitagawa: "Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A"Biophysical Journal. 79. 485-495 (2000)

K.Yamamoto、Y.Mizutani 和 T.Kitakawa：“核糖核酸酶 A 热展开的纳秒温度跳跃和时间分辨拉曼研究”生物物理学杂志。

K. Yamamoto, Y. Mizutani and T. Kitagawa: "Nanosecond Temperature Jump and Time-resolved Raman Study of Thermal Unfolding of Ribonuclease A"Biophysical Journal. (in press). (2000)

K. Yamamoto、Y. Mizutani 和 T. Kitakawa：“核糖核酸酶 A 热展开的纳秒温度跳跃和时间分辨拉曼研究”生物物理学杂志。