Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from Escherichia coli.
The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and required for efficient disulfide bond formation in the bacterial periplasm. The enzyme consists of a thioredoxin-like domain and a second, alpha-helical domain which is inserted into the thioredoxin motif. Reduction of the active-site disulfide in the thioredoxin domain causes a more than 3-fold increase in tryptophan fluorescence. However, both tryptophan residues of the protein, W76 and W126, are not in contact with the disulfide and located in the alpha-helical domain. Analysis of the variants W76F and W126F revealed that the fluorescence of W126 is fully quenched in every redox state of DsbA. W126 is also a sink for nonradiative energy transfer from W76. In oxidized DsbA, W76 is quenched by an intramolecular, dynamic quenching process which involves energy transfer from W76 via F26 to the disulfide. The contributions of the disulfide bridge and the tryptophan residues to the near-UV CD spectra were also quantified. Analysis of the thermodynamic stabilities of the variants W76F and F26L revealed that the interdomain contact between W76 and F26 strongly contributes to the overall stability of DsbA, and selectively stabilizes its oxidized form. The DsbA variant F26L is the most oxidizing disulfide oxidoreductase known so far.
Hennecke
J
J
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland.
Sillen
A
A
Huber-Wunderlich
M
M
Engelborghs
Y
Y
Glockshuber
R
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010641:Phenotype; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; D004220:Disulfides; D012997:Solvents; D014364:Tryptophan; D007535:Isomerases; D019704:Protein Disulfide-Isomerases