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Enhancement of protein stability by an additional disulfide bond designed in human neuroglobin

机译：通过在人类神经球蛋白中设计的附加二硫键增强蛋白质稳定性

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Human neuroglobin (Ngb) forms an intramolecular disulfide bond between Cys46 and Cys55, with a third Cys120 near the protein surface, which is a promising protein model for heme protein design. In order to protect the free Cys120 and to enhance the protein stability, we herein developed a strategy by designing an additional disulfide bond between Cys120 and Cys15 via A15C mutation. The design was supported by molecular modeling, and the formation of Cys15–Cys120 disulfide bond was confirmed experimentally by ESI-MS analysis. Molecular modeling, UV-Vis and CD spectroscopy showed that the additional disulfide bond caused minimal structural alterations of Ngb. Meanwhile, the disulfide bond of Cys15–Cys120 was found to enhance both Gdn·HCl-induced unfolding stability (increased by ～0.64 M) and pH-induced unfolding stability (decreased by ～0.69 pH unit), as compared to those of WT Ngb with a single native disulfide bond of Cys46–Cys55. Moreover, the half denaturation temperature ( T _(m) ) of A15C Ngb was determined to be higher than 100 °C. In addition, the disulfide bond of Cys15–Cys120 has slight effects on protein function, such as an increase in the rate of O _(2) release by ～1.4-fold. This study not only suggests a crucial role of the artificial disulfide in protein stabilization, but also lays the groundwork for further investigation of the structure and function of Ngb, as well as for the design of other functional heme proteins, based on the scaffold of A15C Ngb with an enhanced stability.
机译：人神经球蛋白（Ngb）在Cys46和Cys55之间形成分子内二硫键，并在蛋白质表面附近形成第三个Cys120，这是用于血红素蛋白质设计的有前途的蛋白质模型。为了保护游离的Cys120并增强蛋白质稳定性，我们在本文中开发了一种策略，通过A15C突变在Cys120和Cys15之间设计一个额外的二硫键。该设计得到分子建模的支持，并且通过ESI-MS分析实验确定了Cys15–Cys120二硫键的形成。分子模型，UV-Vis和CD光谱表明，额外的二硫键导致Ngb的结构变化最小。同时，与WT Ngb相比，发现Cys15–Cys120的二硫键既增强了Gdn·HCl诱导的解折叠稳定性（增加了〜0.64 M），又增强了pH诱导的解折叠稳定性（减少了〜0.69 pH单位）。带有一个天然的Cys46–Cys55二硫键。另外，A15C Ngb的半变性温度（T _（m））被确定为高于100℃。此外，Cys15–Cys120的二硫键对蛋白质功能有轻微影响，例如O _（2）释放速率增加〜1.4倍。这项研究不仅表明了人工二硫键在蛋白质稳定中的关键作用，而且还为进一步研究Ngb的结构和功能以及基于A15C支架设计其他功能血红素蛋白奠定了基础。 Ngb具有增强的稳定性。

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