Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One
Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One
复制标题
DOI：
10.3390/biom9060250
复制
发表时间：
2019-06-01
期刊：
影响因子：
5.5
通讯作者：
Kuroda, Yutaka
中科院分区：
文献类型：
--
作者：
Saotome, Tomonori;Yamazaki, Toshio;Kuroda, Yutaka
在实践中，尽管Anfinsen的教条，重组多SS-键合蛋白质的重折叠是众所周知的困难，因为错误折叠的物种与非天然SS-键出现后，其半胱氨酸残基的氧化。另一方面，单SS-键蛋白被认为是简单的重折叠，因为它们的半胱氨酸只有一个SS-键伴侣。在这里，我们报告，登革4包膜蛋白结构域3（DEN 4 ED 3），一个单一的SS-键合蛋白可以不可逆地陷入一个错误折叠的物种，通过形成其唯一的分子内SS-键。在pH值高于约7时，错误折叠物种的溶解度比天然物种低得多，圆二色性测量清楚地表明其二级结构含量与天然物种不同。此外，来自上清液级分的DEN 4 ED 3的异源单量子相关光谱（HSQC）谱中的峰是尖锐的并且分散良好，反映了β-片层的天然结构，而沉淀级分的谱显示出在其中心附近聚集的宽信号，表明没有或几乎没有结构和强烈的聚集倾向。这两个物种具有不同的生物物理性质，可以相互转化成对方只有通过切割和改造的SS-键，强烈表明它们是拓扑不同的。这种现象可能发生在任何单一的SS-键合蛋白质，我们的观察强调需要评估的构象和生物物理性质的细菌产生的治疗性蛋白质，除了他们的化学纯度。
In practice and despite Anfinsen's dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.