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

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

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.
OriginalsprogEngelsk
TidsskriftEMBO Journal
Vol/bind20
Udgave nummer21
Sider (fra-til)5853-5862
Antal sider10
ISSN0261-4189
DOI
StatusUdgivet - 2001
Eksternt udgivetJa

ID: 43973698