Disruption of the disulfide bridge in azurin from Pseudomonas aeruginosa.
In the family of small blue-copper proteins azurins are unique in that they contain a disulfide bridge close to the amino-terminal end. It is situated in the 'south' side of the molecule, about 25 A away from the copper. Site-specific mutagenesis was used to exchange one or both of the cysteines in the bridge for serines in Pseudomonas aeruginosa azurin. In the double mutant Cys3Ser-Cys26Ser the Type 1 Cu is converted into Type 2, and the fluorescence of the single internal tryptophan shows that it becomes exposed to a polar environment. The circular dichroism spectrum indicates a loss of beta-structure. Thus, this mutation prevents the correct folding of the protein and the formation of the metal-binding site. Single mutants, Cys3Ser or Cys26Ser, can at least in part form native-like structures as shown by optical, EPR, fluorescence and CD spectroscopy. The Cys3Ser mutant can form a stable intermolecular disulfide bond which promotes the native conformation of the protein.
Bonander
N
N
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Karlsson
B G
BG
Vänngård
T
T
D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012694:Serine; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D004220:Disulfides; D001400:Azurin; D012694:Serine; D003300:Copper; D003545:Cysteine