Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of cili...Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis. Then 2-p-toluidinylnaphthalene-6- sulfonate (TNS) was used as a fluorescence probe to detect the conformational change of the protein. The results show that the metal- binding capability of the site I of EoCen was lost by the mutation of Asp37→Lys. In comparison the Tb3+-saturated EoCen, the hydrophobic surface of D37K, which is exposed by the binding of Tb3+, has shrunk sharply, suggesting that Asp37 plays an important role in maintaining the proper conformation of EoCen in the presence of Tb3+. Meanwhile, the conditional binding constants of TNS with Tb3+-loaded EoCen and D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38 ±0.25)×105 M-1 and K(Tb3+-D37K-TNS)=(1.16±0.05)×106 M-1.展开更多
文摘Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis. Then 2-p-toluidinylnaphthalene-6- sulfonate (TNS) was used as a fluorescence probe to detect the conformational change of the protein. The results show that the metal- binding capability of the site I of EoCen was lost by the mutation of Asp37→Lys. In comparison the Tb3+-saturated EoCen, the hydrophobic surface of D37K, which is exposed by the binding of Tb3+, has shrunk sharply, suggesting that Asp37 plays an important role in maintaining the proper conformation of EoCen in the presence of Tb3+. Meanwhile, the conditional binding constants of TNS with Tb3+-loaded EoCen and D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38 ±0.25)×105 M-1 and K(Tb3+-D37K-TNS)=(1.16±0.05)×106 M-1.
