Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.
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.
Ignatova
Zoya
Z
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
Gierasch
Lila M
LM
D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011506:Proteins