DEVELOPMENT OF A PRESSURE-JUMP NMR APPARATUS

我们设计并制作了一个压力跳变装置，目的是将其用于压力跳变核磁共振实验。对丙醇的质子核磁共振谱进行了首次压力跳变核磁共振实验。在不到1秒的时间内成功实现了从1500bar到1bar的跃迁，并以3s的间隔跟踪了丙醇光谱随时间的变化。结果表明，在压力跳变后的3秒内，信号几乎完全移位。高压/高场核磁共振技术已被用于研究高压溶液中蛋白质的结构和变性。利用高压池，在400 MHz、1-2000 atm的压力范围内，利用苯基乙炔在7.5-40‰的温度范围内热膨胀，研究了rna酶A （6.2mM,pH1.0,0.15M KCl, in D_2O）的热力学稳定性。展开后的体积增加，δ tav呈负向且与温度相关，从7.5℃时的-10 ml/mol降至37℃时的30ml/mol。我们还发现DELTACp依赖于压力，从1atm时的1.79 kcal/mol K下降到2000 atm时的1.08 kcal/mol K。在工作频率为750MHz的核磁共振光谱仪中安装了在线高压石英池系统。该系统用于检测几种蛋白质的单个质子的压力诱导化学位移变化，这表明结构变化发生在蛋白质折叠的构象内。在溶菌酶中，疏水团簇是优先压缩的部位。在其他小蛋白质中，大多数肽NH基团的氢键被发现在压力下缩短。

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.