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

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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 Å crystal structure of the oxidized protein revealed a disulfide bond‐induced distortion of the β‐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.