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

H. Østergaard, A. Henriksen, Flemming G. Hansen, J.R. Winther

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom 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 languageEnglish
    JournalE M B O Journal
    Volume20
    Issue number21
    Pages (from-to)5853-5862
    ISSN0261-4189
    Publication statusPublished - 2001

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