Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
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Journal | EMBO Journal |
Volume | 20 |
Issue number | 21 |
Pages (from-to) | 5853-5862 |
Number of pages | 10 |
ISSN | 0261-4189 |
DOIs | |
Publication status | Published - 2001 |
Externally published | Yes |
- Crystallography, X-Ray, Cysteine, Cytoplasm, Disulfides, Escherichia coli, Gene Expression, Green Fluorescent Proteins, Luminescent Proteins, Models, Molecular, Mutagenesis, Site-Directed, Oxidation-Reduction, Protein Engineering, Protein Structure, Secondary, Protein Structure, Tertiary, Spectrometry, Fluorescence, Thioredoxins
Research areas
ID: 43973698