Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling
Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling
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DOI：
10.1073/pnas.0304533101
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发表时间：
2004-01-13
影响因子：
11.1
通讯作者：
Gierasch, LM
中科院分区：
文献类型：
--
作者：
Ignatova, Z;Gierasch, LM
表达蛋白的体内荧光标记使得能够直接在细菌细胞中观察其稳定性和聚集。突变哺乳动物细胞视黄酸结合蛋白I（CRABP I）以将序列Cys-Cys-Gly-Pro-Cys-Cys掺入表面暴露的Ω环中，所述序列Cys-Cys-Gly-Pro-Cys-Cys特异性结合双砷荧光素染料（FIAsH）。标记的四-Cys CRABP I的展开伴随着FIAsH荧光的增强，这使得有可能通过在细胞中的尿素滴定来确定这种蛋白质的展开的自由能，并在真实的时间内通过缓慢折叠的、易于聚集的突变体（FIAsH标记的P39 A四-Cys CRABP I）来跟踪包涵体的形成。体内聚集显示出与纯化的体外模型系统中蛋白质聚集的观察结果相似的浓度依赖性表观滞后时间。
In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FIAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FIAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregation-prone mutant (FIAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.