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. 2009 Oct 7;7(19):3969-75.
doi: 10.1039/b907664f. Epub 2009 Jul 31.

Fluorogenic Affinity Label for the Facile, Rapid Imaging of Proteins in Live Cells

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Free PMC article

Fluorogenic Affinity Label for the Facile, Rapid Imaging of Proteins in Live Cells

Rex W Watkins et al. Org Biomol Chem. .
Free PMC article

Erratum in

  • Org Biomol Chem. 2009 Dec 21;7(24):5276

Abstract

Haloalkane dehalogenase (HD) catalyzes the hydrolysis of haloalkanes via a covalent enzyme-substrate intermediate. Fusing a target protein to an HD variant that cannot hydrolyze the intermediate enables labeling of the target protein with a haloalkane in cellulo. The utility of extant probes is hampered, however, by background fluorescence as well as limited membrane permeability. Here, we report on the synthesis and use of a fluorogenic affinity label that, after unmasking by an intracellular esterase, labels an HD variant in cellulo. Labeling is rapid and specific, as expected from the reliance upon enzymic catalysts and the high membrane permeance of the probe both before and after unmasking. Most notably, even high concentrations of the fluorogenic affinity label cause minimal background fluorescence without a need to wash the cells. We envision that such fluorogenic affinity labels, which enlist catalysis by two cellular enzymes, will find utility in pulse-chase experiments, high-content screening, and numerous other protocols.

Figures

Figure 1
Figure 1
Fluorogenic (1 and 2) and fluorescent (3) labels for haloalkane dehalogenase.
Figure 2
Figure 2
Labeling of an HD variant (HaloTag®–NLS3) in live, unwashed U2OS cells at 37 °C as visualized by confocal microscopy. Scale bars = 200 µm. (A) Effect of probe type. Probe (1.0 µM) was incubated with cells for 15 min; (i) 1, (ii) 2, (iii) 3, (iv, v, and vi) overlay with brightfield images. (B) Effect of incubation time. Probe 1 (1.0 µM) was incubated with cells for (i) 10 min, (ii) 15 min, and (iii) 30 min. (C) Effect of probe concentration. Probe 1 was incubated with cells for 15 min at (i) 1 µM, (ii) 10 µM, (iii and iv) overlay with brightfield images. (D) Effect of probe unmasking. Probe (1.0 µM) was incubated with cells for 15 min; (i) unmasked 1, (ii) unmasked 2, and (iii) 3.
Figure 3
Figure 3
Lactone–quinoid equilibrium of 1, 12, and 13.
Figure 4
Figure 4
Effect of dielectric constant on the lactone–quinoid equilibrium of unmasked 1, 12, and 13. Absorption spectra of (A) unmasked 1 (50 µM), (B) 12 (12.5 µM), and (C) 13 (12.5 µM) in mixtures of dioxane and water. (D) Absorption at λmax in the spectra in panels A–C. Values of ε are from ref. .
Scheme 1
Scheme 1
Synthesis of probe 1.

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