DOI: 10.1073/pnas.0304533101. Monitoring protein stability and aggregation<i>in vivo</i>by real-time fluorescent labeling

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

10.1073/pnas.0304533101

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

In vivofluorescent 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). Aggregationin vivodisplayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purifiedin vitromodel systems.

Bibliography

Ignatova, Z., & Gierasch, L. M. (2003). Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling. Proceedings of the National Academy of Sciences, 101(2), 523â528.

Authors 2

Zoya Ignatova (first)
Lila M. Gierasch (additional)

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Dates

Type	When
Created	21 years, 7 months ago (Jan. 13, 2004, 1:28 p.m.)
Deposited	1 year, 7 months ago (Jan. 11, 2024, 5:09 p.m.)
Indexed	4 months ago (April 25, 2025, 3:48 a.m.)
Issued	21 years, 7 months ago (Dec. 30, 2003)
Published	21 years, 7 months ago (Dec. 30, 2003)
Published Online	21 years, 7 months ago (Dec. 30, 2003)
Published Print	21 years, 7 months ago (Jan. 13, 2004)

Funders 0

None

BibTeX

@article{Ignatova_2003, title={Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling}, volume={101}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0304533101}, DOI={10.1073/pnas.0304533101}, number={2}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Ignatova, Zoya and Gierasch, Lila M.}, year={2003}, month=dec, pages={523–528} }

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