摘要
富含甘氨酸的RNA结合蛋白AtGrp7是拟南芥(Arabidopsis thaliana)调节生物钟负反馈回路的组分.在使用常规方法纯化AtGrp7 RRM结构域的初始试验中,观察到烟草蚀纹病毒(TEV)酶切后AtGrp7 RNA识别基序(RRM)结构域的紫外吸收峰为蛋白和杂质的混合信号峰.为解决常规纯化中的杂质问题,对AtGrp7_(1-90)应用了变性-复性两步纯化方法. AtGrp7 RRM结构域的~1H-^(15)N HSQC指纹谱和CS-Rosetta模型结构表明快速稀释重折叠后其结构完全恢复.等温滴定量热法(ITC)和核磁共振(NMR)滴定实验进一步证实,重折叠后AtGrp7_(1-90) RRM结构域具有正确结合RNA/DNA的功能.
The glycine-rich RNA-binding protein,AtGrp7,is a component of a negative feedback loop in the circadian clock regulation of Arabidopsis thaliana.In our initial purification trial of the tobacco etch virus(TEV)-cleaved AtGrp7 RNA recognition motif(RRM)domain with the regular protocol,mixed ultraviolet signals of the target proteins and contaminants were observed.A two-step denaturing-refolding protocol was then tested,trying to solve the problem of impurities.The structure of the AtGrp71-90 RRM domain was fully recovered by quick-dilution refolding,evidenced by the fingerprint 1H-15N HSQC spectrum and CS-Rosetta model structures.Isothermal titration calorimetry(ITC)and NMR titration experiments further confirmed that the RRM domain of AtGrp71-90 had proper functions with regards to RNA/DNA binding.
作者
迟秀娟
乔晓亚
刘颖
刘惠丽
陈雷
王际辉
艾选军
CHI Xiu-juan;QIAO Xiao-ya;LIU Ying;LIU Hui-li;CHEN Lei;WANG Ji-hui;AI Xuan-jun(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China;National Laboratory for Clean Energy,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China;Division of Virology&Immunology,National Center for AIDS/STD Control and Prevention,Beijing 102206,China;State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics,National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics,Chinese Academy of Sciences),Wuhan 430071,China)
出处
《波谱学杂志》
CAS
北大核心
2019年第1期1-14,共14页
Chinese Journal of Magnetic Resonance
基金
The Liaoning Natural Science Foundation(20170520198,20170520043)
Project Supported by the State Key Laboratory ofMagnetic Resonance and Atomic and Molecular Physics(T151601)
