摘要
目的构建参与唇腭部发育的重要蛋白ESCRTⅢ的单分子检测体系。方法分析ESCRTⅢ蛋白的结构,选取不影响结构稳定和纤丝形成的氨基酸进行突变,体外提取突变蛋白检测蛋白的稳定性,吸收光谱检测突变蛋白荧光标记的效率,负染电镜观察突变蛋白形成纤丝的能力,负染电镜观察和离心沉淀分析突变体蛋白形成的纤丝被Vps4解聚的能力。结果选取了ESCRTⅢ蛋白Snf7上的5个位点,K57,G58,N59,G120和D122,分别突变为半胱氨酸,其中K57C突变体蛋白不稳定聚沉,G58C突变体蛋白荧光标记效率低,N59C和G120C突变体蛋白影响纤丝形成,只有D122C突变体蛋白稳定,标记效率高,且标记后的蛋白能正常形成纤丝并被Vps4蛋白解聚。结论筛选出了Snf7 D122C突变体适用于利用单分子技术检测ESCRTⅢ纤丝的解聚动态过程,从而为进一步在细胞水平和体内研究ESCRT系统对腭部发育的作用提供支持。
Objective To construct a single-molecule detection system for ESCRTⅢ, an important proteins involved in the development of lip and palate. Methods The structure of the ESCRTⅢ protein was analyzed, and the amino acids which predicted to have no affection on the structural stability and filament formation were selected for mutation. The mutant proteins were extracted and the stabilities were detected. The efficiency of fluorescence labeling of mutant protein was detected by absorption spectrum. The ability of the mutant protein to form filaments was observed by negative staining electron microscope, and the ability of filament formed by mutant protein to be depolymerized by Vps4 was observed by negative staining electron microscope and analyzed by centrifugal precipitation analysis. Results Five sites on ESCRTⅢ protein Snf7, K57, G58, N59, G120 and D122, were selected to mutate to cysteine. K57C mutant protein was unstable. G58C mutant protein had low fluorescence labeling efficiency. Mutation on N59 and G120 mutant protein affected the formation of filament. Only the D122C mutant protein was stable and the labeling efficiency was high. The labeled protein formed filament that could be depolymerized by Vps4 protein. Conclusions The Snf7 D122C mutant was screened out, which is suitable for further detection of the depolymerization dynamic process of ESCRTⅢ filaments by the single-molecule technique, so as to provide supports for further study of the effect of ESCRT system on development of palate at the cellular level and in vivo.
作者
李瑶
杨梦铱
李琳
陈春来
隋森芳
孙珊
Li Yao;Yang Mengyi;Li Lin;Chen Chunlai;Sui Senfang;Sun Shan(State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China)
出处
《中华口腔正畸学杂志》
2019年第3期152-156,共5页
Chinese Journal of Orthodontics
基金
国家自然科学基金(31670746).
