Bonander N, Karlsson B G, Vänngård T
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Biochim Biophys Acta. 1995 Aug 16;1251(1):48-54. doi: 10.1016/0167-4838(95)00059-4.
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.
在小蓝铜蛋白家族中，天青蛋白是独特的，因为它们在靠近氨基末端处含有一个二硫键。它位于分子的“南侧”，距离铜约25埃。在铜绿假单胞菌天青蛋白中，采用定点诱变将该桥中的一个或两个半胱氨酸替换为丝氨酸。在双突变体Cys3Ser-Cys26Ser中，1型铜转变为2型，单个内部色氨酸的荧光表明它暴露于极性环境中。圆二色光谱表明β结构丧失。因此，这种突变阻止了蛋白质的正确折叠和金属结合位点的形成。如光学、电子顺磁共振、荧光和圆二色光谱所示，单突变体Cys3Ser或Cys26Ser至少可以部分形成类似天然的结构。Cys3Ser突变体可以形成稳定的分子间二硫键，从而促进蛋白质的天然构象。
