Liu Hai-Xiao, Li Lianzhi, Yang Xin-Zhi, Wei Chuan-Wan, Cheng Hui-Min, Gao Shu-Qin, Wen Ge-Bo, Lin Ying-Wu
School of Chemistry and Chemical Engineering, University of South China Hengyang 421001 China
School of Chemistry and Chemical Engineering, Liaocheng University Liaocheng 252059 China.
RSC Adv. 2019 Jan 31;9(8):4172-4179. doi: 10.1039/c8ra10390a. eCollection 2019 Jan 30.
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 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 ( ) 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 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.
人神经球蛋白（Ngb）在半胱氨酸46（Cys46）和半胱氨酸55（Cys55）之间形成分子内二硫键，在蛋白质表面附近还有第三个半胱氨酸120（Cys120），这是血红素蛋白设计中一个很有前景的蛋白质模型。为了保护游离的Cys120并提高蛋白质稳定性，我们在此开发了一种策略，即通过设计Cys120与Cys15之间的额外二硫键（A15C突变）来实现。该设计得到了分子模拟的支持，并且通过电喷雾电离质谱（ESI-MS）分析实验证实了Cys15-Cys120二硫键的形成。分子模拟、紫外可见光谱和圆二色光谱表明，额外的二硫键对Ngb的结构改变最小。同时，与具有单个天然Cys46-Cys55二硫键的野生型Ngb相比，发现Cys15-Cys120二硫键增强了盐酸胍（Gdn·HCl）诱导的去折叠稳定性（增加约0.64 M）和pH诱导的去折叠稳定性（降低约0.69个pH单位）。此外，A15C Ngb的半变性温度（）被确定高于100℃。此外，Cys15-Cys120二硫键对蛋白质功能有轻微影响，例如氧释放速率增加约1.4倍。这项研究不仅表明人工二硫键在蛋白质稳定中起关键作用，还为基于具有增强稳定性的A15C Ngb支架进一步研究Ngb的结构和功能以及设计其他功能性血红素蛋白奠定了基础。
