一种利用荧光蛋白mNeonGreen2鉴定EI24蛋白拓扑结构的方法（英文）

A Method for Identifying The Topology of Etoposide-induced Protein 2.4 Using Split mNeonGreen2

方便且精准地检测跨膜蛋白拓扑结构,尤其是跨膜片段的氨基(N-)和羧基(C-)端的朝向,有利于发现新的蛋白质与蛋白质之间的相互作用,并进一步揭示蛋白质重要的生物学功能.自组装荧光蛋白已被广泛用于观察蛋白质与蛋白质之间的相互作用、标记细胞内源蛋白质并实现mRNA定位的可视化.本文扩展了自组装荧光蛋白的应用,将自组装荧光蛋白mNeonGreen2与定点标记技术相结合,以确定跨膜蛋白的拓扑结构.通过该方法,第一次清楚地证明了EI24的N端和C端均朝向细胞质方向.此外,该方法可用于确定定位于其他细胞器且结构尚未解析的跨膜蛋白的拓扑结构.

Convenient,reliable detection of transmembrane protein topology,especially the orientation of the amino(N-)and carboxyl(C-)termini of a membrane-spanning segment,may aid in identifying protein-protein interactions and clarifying the important biological functions of proteins.Self-complementing split fluorescent proteins have been widely used to image protein-protein interactions,label endogenous proteins and visualize mRNA localization.Here,we expand this toolset and develop an efficient method combining a self-complementing split mNeonGreen2 with site-directed labeling(SSDL)to identify the topology of transmembrane proteins.With SSDL,for the first time,we clearly demonstrate that both the N- and C-termini of etoposide-induced protein 2.4,which localizes in the endoplasmic reticulum,have a cytosolic orientation.This method can be useful for determining the topology of other organelle-based transmembrane proteins that have insufficient structural information.