Abstract
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conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis.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.
Highlights
Centrin is a calcium-binding protein of ~20 kDa, and present in both the pericentriolar material and the centrioles of centrosome
The effect of the mutation on the conformation of wild-type Euplotes octocarinatus centrin (EoCen) was monitored by Far-UV circular dichroism (CD), the Far-UV light CD spectra of EoCen and D37K are highly similar in shape, which means that the mutation of aspartic acid does not disrupt the secondary structure of wild-type EoCen
The mutant D37K had the same mobility as wild type EoCen on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Figure 2(a)), since the molecular weight of D37K nearly did not change after mutation with an apparent molecular mass of 20 kDa, indicating highly purified proteins were obtained
Summary
Centrin is a calcium-binding protein of ~20 kDa, and present in both the pericentriolar material and the centrioles of centrosome. Genetic studies show that centrin is essential to normal cell cycle-dependent duplication and segregation of the microtubule organizing center (MTOC) [1]. Much research has focused on these functions, because abnormal centrosome duplication may lead to chromosomal instability and cancer, an idea supported by discovery of supernumerary abnormal centrosomes in different human tumor cells [4,5,6]. Conformational changes are intrinsic to the function of a variety of proteins. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS) has been extensively used in the conformational change of centrin induced by metal ions [10,11,12] Conformational changes are intrinsic to the function of a variety of proteins. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS) has been extensively used in the conformational change of centrin induced by metal ions [10,11,12]
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