DEVELOPMENT OF A PRESSURE-JUMP NMR APPARATUS

我们设计并制作了一台压力跳跃装置，用于压力跳跃核磁共振实验。首次对丙醇的质子核磁共振谱进行了压跃核磁共振实验。从1500bar到1bar的跃迁发生在不到1秒的时间内，丙醇的光谱随时间变化的间隔约为3s。高压/高场核磁共振技术被发展起来并用于研究高压下蛋白质在溶液中的结构和变性。在1-2000atm的压力范围内，用高压室研究了核糖核酸酶A(6.2 mM，pH1.0，0.15MKCl，在D_2O中)在1-2000atm压力范围内的热力学稳定性。在7.540ﾟC温度范围内，苯乙炔的热膨胀引起的体积增加是负的，且与温度有关，从7.5ﾟC时的-10ml/ﾟ下降到37℃时的-30ml/mol。从1大气压下的1.79千卡/摩尔K下降到2000大气压下的1.08大卡/摩尔K。在750 MHz频段的核磁共振谱仪上安装了在线高压石英池系统。该系统被用来检测压力诱导的几种蛋白质单个质子的化学位移变化，这表明蛋白质的折叠构象发生了结构变化。在溶菌酶中，疏水簇是优先压缩的位置。在其他小蛋白中，多肽NH基团的大多数氢键被发现随着压力的增加而缩短。

We have designed and constructed a pressure-jump apparatus with a purpose of using it in pressure-jump NMR experiments. The first pressure-jump NMR experiment was performed for proton NMR spectrum of propanol. The jump was successfully attained from 1500 bar to 1 bar much less than a second, and the spectral change of propanol were followed with time at about 3s interval. The result showed that the signals shifted almost completely at least within 3s upon the pressure jump.High pressure/high field NMR techniques have been developed and used for studying structure and denaturation of proteins in solution under high pressure. Thermodynamic stability of ribonuclease A (6.2mM,pH1.0,0.15M KCl, in D_2O) has been studied by ^1H NMR at 400 MHz in the pressure range of 1-2000 atm by using high pressure cell due to thermal expansion of phenylacetylene in the temperature range of 7.5-40ﾟC.The volume increase upon unfolding, DELTAV,was negative and temperature-dependent, decreasing from -10 ml/mol at 7.5ﾟC to-30ml/mol at 37ﾟC.We also found that DELTACp is dependent on pressure, decreasing from 1.79 kcal/mol K at 1 atm to 1.08 kcal/mol K at 2000 atm. An on-line high pressure quartz cell system was installed into an NMR spectrometer operating at 750MHz. This system was used to detect pressure-induced chemical shift changes of individual protons of several proteins, which demonstrated that structural changes take place within the folded conformer of a protein. In lysozyme, the hydrophobic cluster was the site of preferential compression. In other small proteins, most hydrogen bonds of the peptide NH groups were found to be shortened by pressure.