Urea-induced unfolding of the alpha subunit of tryptophan synthase: one-dimensional proton NMR evidence for residual structure near histidine-92 at high denaturant concentration.
Urea-induced unfolding of the alpha subunit of tryptophan synthase: one-dimensional proton NMR evidence for residual structure near histidine-92 at high denaturant concentration.
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DOI：
10.1021/bi00213a031
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发表时间：
1993-12
期刊：
影响因子：
2.9
通讯作者：
G. Saab-Rincón;C. Froebe;C. Matthews
中科院分区：
文献类型：
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作者：
G. Saab-Rincón;C. Froebe;C. Matthews
通过使用 1D NMR 光谱检查四个组氨酸残基的 C ε 质子的化学位移和峰面积来监测尿素诱导的色氨酸合酶 α 亚基的展开反应。在由酪氨酸吸光度和远紫外圆二色光谱定义的天然基线区域中，组氨酸-146 似乎在变性剂浓度增加时经历快速的局部去折叠反应。由于天然形式转化为之前检测到的 2 和 3 M 尿素之间的稳定中间体 [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784]，氨基折叠单元中的组氨酸-92 和 -146（残基 1-188）以及羧基折叠单元中的组氨酸-195 和 -244（残基） 189-268）所有人都经历了环境的变化，这种变化在核磁共振时间尺度上是缓慢的。当考虑到涉及 His-244 的分子间缔合过程时，该中间体随后转化为新检测到的、稳定的、部分折叠的、以 5 M 尿素填充的形式，在 25°C 下似乎对任何组氨酸都没有影响。引人注目的是，观察到仅涉及 His-92 的缓慢交换过程从 5 M 尿素开始，其中通过吸光度或远紫外圆二色光谱监测的展开转变基本完成。当尿素浓度增加到 8 M 时，这种残留的三级结构以协同方式展开。
The urea-induced unfolding reaction of the alpha subunit of tryptophan synthase was monitored by examining the chemical shifts and peak areas of the C epsilon protons of the four histidine residues with 1D NMR spectroscopy. In a native base-line region defined by tyrosine absorbance and far-UV circular dichroism spectroscopy, histidine-146 appears to undergo a rapid, local unfolding reaction at increasing denaturant concentrations. As the native form is converted to a previously detected stable intermediate between 2 and 3 M urea [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784], histidines-92 and -146 in the amino folding unit (residues 1-188) and histidines-195 and -244 in the carboxy folding unit (residues 189-268) all experience a change in their environments which is slow on the NMR time scale. The subsequent conversion of this intermediate to a newly detected, stable, partially folded form populated at 5 M urea appears to have no effect on any of the histidines at 25 degrees C when an intermolecular association process involving His-244 is taken into account. Strikingly, a slow exchange process involving only His-92 is observed to begin at 5 M urea where the unfolding transitions monitored by absorbance or far-UV circular dichroism spectroscopy are essentially complete. This residual tertiary structure unfolds in a cooperative fashion as the urea concentration is increased to 8 M.