Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.
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.
Ostergaard
H
H
Section of Molecular Microbiology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark.
Henriksen
A
A
Hansen
F G
FG
Winther
J R
JR
D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D003545:Cysteine