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.

Fig. 1.

(A) Backbone structure of CRABP I (rendered by using insight II from PBD file 1CBI) showing the point of insertion of the tetra-Cys motif between strands β7 and β8 in the omega loop (circled) and the location of P39, at the end of the second α-helix. (B) CD spectra comparing CRABP I WT*, tetra-Cys CRABP I, P39A tetra-Cys CRABP I, and FlAsH-labeled tetra-Cys CRABP I. (C) fluorescence of FlAsH-labeled tetra-Cys CRABP I (excitation 500 nm) in its native folded state and denatured by 8 M urea (unfolded), showing the hyperfluorescence of the unfolded state.

Fig. 2.

(A) FlAsH labeling of tetra-Cys CRABP I and P39A tetra-Cys CRABP I in E. coli cells. Before IPTG induction of protein expression, cells were treated with dye, either after lysozyme pretreatment (solid lines) or not (dashed lines). Spectra shown were obtained 4 h after induction. (B) Urea denaturation curves monitored using fluorescence of FlAsH-labeled tetra-Cys CRABP I (excitation 500 nm, emission 530). For the in vivo curve, urea was added to the indicated concentration 2 h after induction of protein expression in cells preloaded with FlAsH.

Fig. 3.

(A) Time course of FlAsH fluorescence at 530 nm after lysozyme pretreatment, loading with dye, and IPTG induction of protein expression at time 0 (tetra-Cys CRABP I in red, P39A tetra-Cys CRABP I in blue; filled symbols). Cell density (OD₆₀₀) is shown in open symbols in the same colors. (B) Time evolution of P39A tetra-Cys CRABP I protein distribution between soluble and insoluble fractions from cell fractionation (note that all of the tetra-Cys CRABP I remains soluble throughout the same time course). (C) Fluorescence microscopy of cells at 60 min (Left) or 240 min (Right) postinduction showing increasing number of highly fluorescent inclusion bodies with polar distribution. (D) Effect of chloramphenicol (Chlm) treatment at a concentration that reduces translation (and therefore concentration of expressed protein) on the time course of FlAsH-labeled P39A tetra-Cys CRABP I fluorescence. Note the longer apparent lag time in the presence of chloramphenicol (⋄) before the striking increase in fluorescence of FlAsH-labeled P39A tetra-Cys CRABP I.