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, 21 (20), 6067-70

Localization of GroEL Determined by in Vivo Incorporation of a Fluorescent Amino Acid

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Localization of GroEL Determined by in Vivo Incorporation of a Fluorescent Amino Acid

Godefroid Charbon et al. Bioorg Med Chem Lett.

Abstract

The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.

Figures

Figure 1
Figure 1. GroEL129CouAA biochemical characterization
a) The efficiency of amber suppression was tested by growing cells harboring plasmids for GroEL129CouAA, MjtRNA, and CouRS expression in the absence or presence of coumarin amino acid, and by analyzing the whole cell lysates by SDS PAGE. The left panel shows the Coomassie- stained gel. The right panel shows GroEL129CouAA excited using a 305 nm transilluminator. b) The rate of ATP hydrolysis was measured using a standard malachite green-based assay using 1 μM chaperonin tetradecamer and 1 mM ATP. Displayed is a plot of the amount of inorganic phosphate released as a function of time. c) The ability to refold MDH, a stringent GroEL substrate, was measured. The percentage of MDH activity relative to a native MDH control is plotted as a function of time.
Figure 2
Figure 2. GroEL129CouAA fluorescence characterization and fluorescence recovery after photobleaching
a) Incorporation of the coumarin amino acid was analyzed using fluorescence microscopy. The upper panels show fluorescence using a DAPI filter set and the lower panels show DIC images. The times post-induction are shown beneath the images. b)-d) The dynamics of GroEL were measured using FRAP. b) Single cell FRAP images pre-bleaching, immediately post-bleaching, and 5.6 seconds post-bleaching (5.6). c) A plot of cellular fluorescence intensity corrected for photobleaching during acquisition of images. d) Fluorescence intensity was plotted as a function of time and fit to a single exponential curve to calculate the rate of fluorescence recovery (t1/2 = 2.2 sec).
Figure 3
Figure 3. GroEL remains diffuse after all tested insults
Fluorescence images of cells growing at 45°C (a), in the presence of 100 μg/ml ampicillin and 0.5 M NaCl (b), and in hypertonic solution (0.5 M NaCl) (c).

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