Joint research on structural fluctuations in proteins-Studied by hydrogen isotope exchange and high pressure NMR

采用日本神户大学设计建造的在线高压高分辨核磁共振系统(750 MHz×1H)，在1~2000bar压力范围内研究了压力对水溶液中碱性胰酶抑制物(BPTI)的影响。15N标记BPTI由明尼苏达大学G.Woodward教授制备，13C标记BPTI由罗格斯大学G.Montelione教授制备，常规BPTI样品购自Sigma Chem。Co.1。在压力下测量了15N/1H的二维谱，发现酰胺基团的1H(平均0.075ppm/2kbar)和15N信号(平均0.47ppm/2kbar)有明显的低场位移。这些变化反映了NH-0=C或NH-水氢键的缩短。酰胺氮之间15N压力位移的显著差异(0*1.5ppm/2kbar)表明，15N压力位移也反映了phi和psi角随压力的变化，给出了多肽主干结构在压力下特定位置构象变化的量度。这些变化在螺旋和环区明显大于在b片区。从质子NOE测量中发现，压缩在蛋白质基质中不均匀发生；在BPTI的酶结合片段中压缩很大，这表明该片段具有构象灵活性。15N自旋弛豫测量的初步结果表明，在2000巴的范围内，压力不会显著改变BPTT的快速内部运动。还测量了压力引起的~(13)C化学位移，并对其进行了分析。鉴于上述一系列成功的实验，我们没有对氢交换实验进行详细的劝说。

The on-line high pressure high resolution NMR system (750 MHz for 1H), which was designed and constructed in Kobe University, was used to investigate the effect of pressure on basic pancreatic trypsin inhibitor (BPTI) in aqueous solution in the pressure range between 1 and 2000 bar.The 15N-labeled BPTI was prepared by Prof. G.Woodward, Univ.Minnesota, and the 13C-labeled BPTI was prepared by Prof.G.Montelione, Rutgers Univ., while regular BPTI samples were purchased from Sigma Chem. Co.1. The 15N/1H two-dimensional spectra were measured under pressure, which showed distinctive low field shifts for 1H (average 0.075ppm/2kbar) and 15N signals (average 0.47ppm/2kbar) of amide groups. These shifts reflected shortening of NH---0=C or NH---water hydrogen bonds.2. A significant variation in 15N pressure shifts among amide nitrogens (0*1.5ppm/2kbar) indicates that 15N pressure shift also reflects changes in phi and psi angles with pressure, giving a measure of site-specific conformational changes of a polypeptide backbone structure by pressure. These changes are apparently larger in helices and loop regions than in b sheet.3. From proton NOE measurements, it was disclosed that compression occurs non-uniformly within the protein matrix ; compression was found to be large in the enzyme-binding segment of BPTI, suggesting conformational flexibility in this segment.4. Preliminary results of 15N spin relaxation measurements showed that pressure does not significantly alter the rapid internal motions of BPTT within the range of 2000 bar.5. Pressure-induced 13C chemical shifts were also measured, the analysis of which is being made.6. In view of a body of successful experiments stated above, we did not persue the hydrogen exchange experiments in detail.