通过实时荧光标记在体内监测蛋白质稳定性和聚集情况。
Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.
作者信息
Ignatova Zoya, Gierasch Lila M
机构信息
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
出版信息
Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):523-8. doi: 10.1073/pnas.0304533101. Epub 2003 Dec 30.
Abstract
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 (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH 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, aggregationprone mutant (FlAsH-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.
摘要
对表达蛋白进行体内荧光标记，使得能够直接在细菌细胞中观察其稳定性和聚集情况。将哺乳动物细胞视黄酸结合蛋白I（CRABP I）进行突变，使其在表面暴露的ω环中掺入序列Cys-Cys-Gly-Pro-Cys-Cys，该序列可特异性结合双砷荧光素染料（FlAsH）。标记的四半胱氨酸CRABP I的解折叠伴随着FlAsH荧光增强，这使得通过细胞内尿素滴定来确定该蛋白的解折叠自由能，并实时跟踪由一个易于缓慢折叠和聚集的突变体（FlAsH标记的P39A四半胱氨酸CRABP I）形成包涵体成为可能。体内聚集表现出浓度依赖性的明显延迟时间，这与在纯化的体外模型系统中观察到的蛋白质聚集情况相似。
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