Unknown

Dataset Information

0

Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.


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.

SUBMITTER: Ignatova Z 

PROVIDER: S-EPMC327180 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

altmetric image

Publications

Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.

Ignatova Zoya Z   Gierasch Lila M LM  

Proceedings of the National Academy of Sciences of the United States of America 20031230 2


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  ...[more]

Similar Datasets

| S-EPMC5812636 | biostudies-literature
| S-EPMC7596860 | biostudies-literature
| S-EPMC4521985 | biostudies-literature
| S-EPMC2935039 | biostudies-literature
| S-EPMC8088336 | biostudies-literature
| S-EPMC4051779 | biostudies-literature
| S-EPMC10991154 | biostudies-literature
| S-EPMC4814290 | biostudies-literature
| S-EPMC6707254 | biostudies-literature
| S-EPMC5460034 | biostudies-other