A method for direct measurement of protein stability in vivo

Abstract

The stability of proteins is tuned by evolution to enable them to perform their cellular functions for the success of an organism. Yet, most of the arsenal of biophysical techniques at our disposal to characterize the thermodynamic stability of proteins is limited to in vitro samples. We describe an approach that we have developed to observe a protein directly in a cell and to monitor a fluorescence signal that reports the unfolding transition of the protein, yielding quantitatively interpretable stability data in vivo. The method is based on incorporation of structurally nonperturbing, specific binding motifs for a bis-arsenical fluorescein derivative in sites that result in dye fluorescence differences between the folded and unfolded states of the protein under study. This fluorescence labeling approach makes possible the determination of thermodynamic stability by direct urea titration in Escherichia coli cells. The specific case study we describe was carried out on the predominantly beta-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein (CRABP), expressed in E. coli.

Fig. 7.1

Urea titrations of FlAsH-labeled protein in vivo as a function of the incubation time: (A) tetra-Cys CRABP and (B) P39A tetra-Cys CRABP, after sample incubation for the indicated times in urea, monitored by FlAsH fluorescence. (Reproduced from Ref. (12) with permission from Willey InterScience.)

Fig. 7.2

Cell viability. Change in the viability of the host cells as a function of the time of incubation in 0 M, 1.5 M, and 3 M urea. The viability was quantified by measuring the number of cells capable of forming colonies (left axis, closed circles) and optical density at 600 nm (right axis, open symbols).

Fig. 7.3

Fluorescence microscopy images showing uniformly distributed fluorescence of tetra-Cys CRABP (180 min after induction) and hyperfluorescent dense aggregates of P39A tetra-Cys CRABP at the poles of the cells (at 240 min after induction). Hyperfluorescent impurities in the extracellular medium are marked by an arrow.

Fig. 7.4

Urea titrations of FlAsH-labeled protein in vitro as a function of the incubation time (monitored by Trp fluorescence): (A) tetra-Cys CRABP and (B) P39A tetra-Cys CRABP. The actual urea concentration was determined by measuring the refractive index, and data are curve-fit to a two-state model. (Reproduced from Ref. (12) with permission from Willey InterScience.)

Fig. 7.5

Reversibility of the in vivo urea titrations. FlAsH-labeled tetra-Cys CRABP-expressing cells were treated with 3 M urea for 75 min, and then refolding was initiated by dilution of aliquots into fresh LB medium containing 100 μg/mL ampicillin. After incubation of the cells for 60 min, the extent of return of the FlAsH signal, as a measure of refolding, was monitored by FlAsH fluorescence (open symbols). A urea melt of FlAsH-labeled tetra-Cys CRABP incubated for 75 min is given for comparison (closed symbols).