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

EMBO J. 2001 Nov 1;20(21):5853-62. doi: 10.1093/emboj/20.21.5853.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Cysteine / genetics
  • Cysteine / metabolism
  • Cytoplasm / metabolism
  • Disulfides / chemistry
  • Disulfides / metabolism*
  • Escherichia coli / metabolism
  • Gene Expression
  • Green Fluorescent Proteins
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Protein Engineering / methods*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Spectrometry, Fluorescence
  • Thioredoxins / metabolism

Substances

  • Disulfides
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Thioredoxins
  • Cysteine

Associated data

  • PDB/1H6R