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

AUTOR(ES)

Østergaard, Henrik

FONTE

Oxford University Press

RESUMO

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.

Documentos Relacionados

• Shedding light on the dark and weakly fluorescent states of green fluorescent proteins
• Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register.
• Intramolecular crosslinking of tropomyosin via disulfide bond formation: Evidence for chain register
• Coupling of protein antigens to erythrocytes through disulfide bond formation: preparation of stable and sensitive target cells for immune hemolysis.
• Redox Regulation of the Human Dual Specificity Phosphatase YVH1 through Disulfide Bond Formation*