13C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)
13C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)
复制标题
牛胰蛋白酶抑制剂（BPTI）二硫键异构化的13C-NMR研究
DOI：
10.1007/s10858-016-0055-8
复制
发表时间：
2016
期刊：
影响因子：
2.7
通讯作者：
Kainosho M
中科院分区：
文献类型：
--
作者：
Takeda M;Miyanoiri Y;Terauchi T;Kainosho M
二硫键的构象异构化与蛋白质的动力学和功能方面相关。本文通过对牛胰胰蛋白酶抑制剂（BPTI）Cys碳峰的核磁共振（NMR）线形分析，探讨了BPTI中Cys 14-Cys 38二硫键的构象异构化。在这种方法中，1D 13 C光谱记录在小的温度间隔的BPTI样品选择性标记的位点特异性13 C-富集的Cys，和记录的峰显示在温度的顺序后，光谱标度归一化为碳峰。在轮廓线的形状，交换加宽，随温度变化的表现为Cys 14和Cys 38的碳峰。据报道，Cys 14-Cys 38二硫键存在于高填充状态（M）和两个低填充状态（mc 14和mc 38）之间的平衡。与三位点交换模型一致，观察到Cys 14 α-碳峰的两个过程引起的两相交换增宽。当交换速率等于平衡位点之间的化学位移差（Hz）时，交换展宽最大化，通过碳线形轮廓获得了用于建立13 C弛豫分散实验条件的半定量信息。对于c ~（38）异构化反应，在低温下通过交换实验，从主峰中分辨出次态β位的~ 1H-~（13）C信号。
Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D13C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically13C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (mc14andmc38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for13C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to themc38isomerization, the1H-13C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.