The (not completely irreversible) population of a misfolded state of cytochrome c under folding conditions.
The (not completely irreversible) population of a misfolded state of cytochrome c under folding conditions.
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DOI：
10.1021/bi301586e
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发表时间：
2013-02
期刊：
影响因子：
2.9
通讯作者：
Jonathan B. Soffer;E. Fradkin;Leah A. Pandiscia;R. Schweitzer‐Stenner
中科院分区：
文献类型：
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作者：
Jonathan B. Soffer;E. Fradkin;Leah A. Pandiscia;R. Schweitzer‐Stenner
本文报道了马心铁细胞色素c在碱性pH为11.5的溶液中作用1周后，发现了一种(偏)稳定的部分展开状态。此后，当将溶液调整为在中性pH下促进折叠的条件时，蛋白质的二级和三级结构没有发生任何可检测到的变化。光谱数据表明，错误折叠的蛋白质显示出一个六配位的低自旋状态，可能的配体是羟基离子。在pH 6以下，出现了一个新的配位态，具有血红素铁的五配位量子混合态的光谱特征。凝胶电泳法显示，在亚毫摩尔浓度下，可溶二聚体和三聚体大量形成，而在较低的微摩尔浓度时，单体占主导地位。紫外圆二色谱表明，氧化单体是预熔融的球状到球状，有相当一部分的二级(螺旋)结构让人想起碱性状态V。低聚物含有更多的螺旋结构，这表明结构域交换是其形成的根本机制。亚毫米级单体和低聚物混合物中的很大一部分经历了血红素铁的还原。它对pH的依赖表明它耦合到一个质子转移过程。综上所述，我们的数据表明，铁细胞色素c部分未折叠，其光谱特征使人想起最近发现的碱性异构体V(B)，它在折叠条件下通过将蛋白质暴露在非常碱性的pH中很长一段时间而稳定下来。
This paper reports the discovery of a (meta)stable partially unfolded state of horse heart ferricytochrome c that was obtained after exposing the protein to a solution with an alkaline pH of 11.5 for 1 week. Thereafter, the protein did not undergo any detectable change in its secondary and tertiary structure upon adjusting the solution to folding promoting conditions at neutral pH. Spectroscopic data suggest that the misfolded protein exhibits a hexacoordinated low-spin state with a hydroxyl ion as the likely ligand. Below pH 6, a new ligation state emerges with the spectroscopic characteristics of a pentacoordinated quantum mixed state of the heme iron. Gel electrophoresis revealed substantial formation of soluble dimers and trimers at submillimolar concentrations, whereas monomers were dominant at lower, micromolar concentrations. Ultraviolet circular dichroism spectra indicate that oxidized monomers are pre-molten globule to globule-like with a substantial fraction of secondary (helical) structure reminiscent of alkaline state V. The oligomers contain even more helical structure, which suggests domain swapping as the underlying mechanism of their formation. A substantial fraction of the submillimolar mixture of monomers and oligomers underwent a reduction of the heme iron. Its dependence on pH suggests the coupling to a proton transfer process. Altogether, our data indicate a partially unfolded ferricytochrome c conformation with spectroscopic characteristics reminiscent of the recently discovered alkaline isomer V(b), which is stabilized under folding conditions by exposing the protein to a very alkaline pH for an extended period of time.