期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 101, 期 2, 页码 523-528出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0304533101
关键词
protein aggregation; protein folding; fluorescence
资金
- NIGMS NIH HHS [GM27616, R01 GM027616] Funding Source: Medline
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.
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