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EMBO J. 2001 Nov 1;20(21):5853-62.

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

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  • 1Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark.

Abstract

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

PMID:
11689426
[PubMed - indexed for MEDLINE]
PMCID:
PMC125700
Free PMC Article
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