Developement and Application of High Pressure Multi-Dimensional NMR Spectroscopy

本课题的主要研究成果如下：（1）高压多维核磁共振波谱技术的发展。我们尝试了在高压下（高达2000巴）测量血红素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，通过使用玻璃毛细管作为样品管，需要非常高的样品浓度（超过10 mM）和40-60小时的积累来获得2D NMR谱。虽然玻璃毛细管在NMR探针中的检测线圈下方需要足够的空间，但我们的探针的底部部分用于温度控制单元，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修改使用玻璃毛细管。（2）高压激光闪光光解在血红素蛋白动力学性质研究中的应用。由于压力被认为是扰动蛋白质结构的热涨落，我们研究了热涨落对血红素蛋白动力学性质的影响。我们在这个研究项目中关注的动力学性质之一是血红素蛋白中的配体结合。突变肌红蛋白的系统组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的高疏水环境在控制配体结合中起关键作用。另一个动力学性质是血红素蛋白中的电子转移反应。通过对高压下血红素蛋白（肌红蛋白）电子转移反应速率的测定，指出蛋白质结构中的热涨落对电子转移反应的途径和自由能差有很大的影响，而热涨落是蛋白质电子转移分子机制的关键因素之一。

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.