Inoue K, Yamada H, Imoto T, Akasaka K
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Japan.
J Biomol NMR. 1998 Nov;12(4):535-41. doi: 10.1023/a:1008374109437.
The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40 degrees C. Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0-0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H alpha signals of the residues in the beta-sheet showed a negative correlation with the 'folding' shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the beta-sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the 'folding' shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggest that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.
利用一台在750兆赫兹下运行的在线可变压力核磁共振系统，在水性环境（95% 1H₂O/5% 2H₂O，pH 2.0）中研究了压力对匙羹藤蛋白（一种来自匙羹藤的含35个残基的球状蛋白）结构的影响。在40摄氏度下，测量了二维全相关谱（TOCSY）和核欧沃豪斯效应谱（NOESY）随压力在1至2000巴之间的变化。实际上，匙羹藤蛋白的几乎所有质子信号都随压力发生了一些位移，表明整个蛋白质结构对压力有响应并因压力而改变。大多数酰胺质子随压力呈现出不同程度的低场位移，即在2000巴时为0至0.2 ppm，平均为0.051 ppm，这表明它们参与了氢键形成，并且这些氢键因压力而不同程度地缩短。还证实了暴露于溶剂（水）中的酰胺质子的化学位移比与羰基内部形成氢键的质子对压力更敏感。β-折叠中残基的Hα信号的压力诱导位移与“折叠”位移（1巴时的位移与无规卷曲位移之差）呈负相关，表明β-折叠的主链扭转角因压力而略有改变。侧链质子也观察到了显著的压力诱导位移（但不超过“折叠”位移的10%），这表明匙羹藤蛋白的三级结构也受到压力影响。最后，压力诱导位移的线性表明，在1至2000巴的压力范围内，匙羹藤蛋白的压缩性是不变的。
