Developement and Application of High Pressure Multi-Dimensional NMR Spectroscopy

高压多维核磁共振波谱技术的发展与应用

• 批准号: 07558215
• 负责人: MORISHIMA Isao
• 金额: $ 1.98万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558215/
• 关键词: High Pressure Muiti-Nuclear NMR; High Presure Flash Photolysis; Hemoproteins; Electron Transfer in Proteins

The primary results in this research project are as follows :(1)Development of High Pressure Multi-Dimensional NMR Spectroscopy. We have tried to measure 2-dimensional proton NMR spectrum of hemoprotein under high pressure (up to 2000 bar). Our preliminary measurement in normal pressure has revealed that the very high sample concentration (more than 10 mM) and 40-60 hours accumulation would be required to obtain the 2D NMR spectrum by using a glass capillary as sample tube. Although the glass capillary needs enough space below the detection coil in the NMR probe, the bottom part of the our probe was used for the temperature control unit, which severely limits the size of the glass capillary. We final concluded that our NMR probe must be modified to use the glass capillary.(2)Application of High Pressure Laser Flash Photolysis to Dynamic Properties of Hemoproteins. Since pressure has been considered to perturb thermal fluctuation in protein structure, we examined the effects of the thermal fluctuation on dynamic properties of hemoprotein. One of the dynamic properties we focused on in this research project was the ligand binding in hemoproteins. Systematic combination of mutant myoglobins and kinetic measurement under high pressure has clearly shown that the some of the hydrophobic amino acid residues play a key role in controlling the ligand binding by maintaining the high hydrophobic environments in the heme pocket. Another dynamic property is the electron transfer reaction in hemoproteins. Basied on the measurements of the reaction rates for electron transfer in hemoprotein (myoglobin) under high pressure, we can pointed out that the electron transfer pathway and free energy difference for the reaction would highly depend on the thermal fluctuation in protein structure, and the thermal fluctuation is one of the critical factors of the molecular mechanism for the electron transfer in proteins.

本课题的主要研究成果如下：(1)高压多维核磁共振波谱的发展。我们尝试在高压(高达2000bar)下测量了血红蛋白的二维质子核磁共振谱。我们在常压下的初步测量表明，使用玻璃毛细管作为样品管，需要很高的样品浓度(大于10 mm)和40-60小时的积累才能获得2D核磁共振谱。虽然在核磁共振探头中，玻璃毛细管需要在探测线圈下方有足够的空间，但我们的探头底部被用作温度控制单元，这严重限制了玻璃毛细管的尺寸。我们最终得出结论，我们的核磁共振探针必须经过修饰才能使用玻璃毛细管。(2)高压激光闪光光解技术在血液蛋白动态性质研究中的应用。由于压力被认为会扰乱蛋白质结构中的热波动，我们研究了热波动对血红素蛋白动态性质的影响。我们在这项研究项目中关注的动态性质之一是血红素蛋白中的配基结合。突变肌红蛋白的系统结合和高压下的动力学测量清楚地表明，一些疏水氨基酸残基通过维持血红素口袋中高度疏水的环境，在控制配体结合方面发挥了关键作用。另一个动力学性质是血红素蛋白中的电子转移反应。通过对高压下血红蛋白(肌红蛋白)中电子转移反应速率的测量，指出电子转移途径和反应的自由能差在很大程度上取决于蛋白质结构的热涨落，而热涨落是蛋白质电子转移分子机制的关键因素之一。

项目成果

Uchida, T: "The Effects of the Intermolecular Disulfide Bond on Ligand Binding Dynamics in Myoglobin" Biochemistry. 36. 324-332 (1997)

Uchida, T：“分子间二硫键对肌红蛋白配体结合动力学的影响”生物化学。

Sugiyama, Y., Takahashi, S., Ishimori, K., Morishima, I.: "Pressure Effects on Electron Transfer Rates in Zinc/Ruthenium Modified Myoglobins" J.Am.Chem.Soc.119. 9582-9583 (1997)

Sugiyama, Y.、Takahashi, S.、Ishimori, K.、Morishima, I.：“压力对锌/钌修饰肌红蛋白中电子转移率的影响”J.Am.Chem.Soc.119。