TY - JOUR

T1 - Shedding light on disulfide bond formation

T2 - engineering a redox switch in green fluorescent protein

AU - Østergaard, Henrik

AU - Henriksen, Anette

AU - Hansen, Flemming G.

AU - Winther, Jakob R.

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

KW - Crystallography, X-Ray

KW - Cysteine

KW - Cytoplasm

KW - Disulfides

KW - Escherichia coli

KW - Gene Expression

KW - Green Fluorescent Proteins

KW - Luminescent Proteins

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Oxidation-Reduction

KW - Protein Engineering

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Spectrometry, Fluorescence

KW - Thioredoxins

U2 - 10.1093/emboj/20.21.5853

DO - 10.1093/emboj/20.21.5853

M3 - Journal article

C2 - 11689426

VL - 20

SP - 5853

EP - 5862

JO - E M B O Journal

JF - E M B O Journal

SN - 0261-4189

IS - 21

ER -