DEVELOPMENT OF A PRESSURE-JUMP NMR APPARATUS

我们设计并制作了一个用于压力跃变核磁共振实验的压力跃变装置。对丙醇的质子核磁共振谱进行了首次压力跃变核磁共振实验。成功地实现了从1500巴到1巴的跳跃，远小于1秒，并且以约3秒的间隔跟踪丙醇的光谱随时间的变化。高压/高场核磁共振技术已经发展起来并用于研究高压下溶液中蛋白质的结构和变性。核糖核酸酶A的热力学稳定性本文用高压盒在1-2000 atm压力范围内，用~ 1H NMR研究了苯乙炔（6.2mM，pH1.0，0.15MKCl，溶于D_2O）在7.5-40 ℃温度范围内的热膨胀，在400 MHz下的去折叠体积增量Δ V为负值，并与温度有关，从7.5 ℃时的-10 ml/mol下降到37 ℃时的-30 ml/mol。我们还发现DELTACP依赖于压力，从1 atm时的1.79 kcal/mol K下降到2000 atm时的1.08 kcal/mol K。将在线高压石英池系统安装到在750 MHz下操作的NMR谱仪中。该系统被用来检测压力诱导的化学位移变化的个别质子的几种蛋白质，这表明结构的变化发生在折叠构象的蛋白质。在溶菌酶中，疏水团簇是优先压缩的位点。在其他小蛋白质中，发现肽NH基团的大多数氢键被压力缩短。

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.