| Centrins are a molecular mass of 20 kD acid proteins from the highly conserved Ca2+ binding EF-hand superfamily. It exits in the centrosomal structures (basal body or spindle pole body) in a wide range of organisms, ranging from algal, yeast and protozoa to higher plants and humans. It involves in centrosome replication and separation, fiber contraction, and flagellar excision and so on.Comparative sequence analysis suggests that Ciliate Euplotes octocarinatus centrins (EoCen) have two relatively independent domains (N-terminal and C-terminal). Each domain consists of two EF-hand (helix-loop-helix) motifs and possesses two potential Ca2+ -binding sites with varying affinities that are determined largely by the amino acid sequence of the binding loops. To explore the importance of Asp37 and Asp73 in the loop of N-terminal of EoCen, the mutants of N-D37K and N-D73K (D is aspartic acid, and K is lysine) were obtained by molecular biological methods.Based on D37K, the mutant of N-D37K was obtained using PCR technology, and the mutant of N-D73K was obtained by site-directed mutagenesis. The sequence of the clones was confirmed by commercial company. Recombinant plasmids were transformed in E.coli BL21 (DE3), and then the fusion expression of the mutants was performed by the induction of IPTG. Fusion protein was cut by PPase and was purified by GST affinity chromatography. At the same time, the wide-type EoCen and the truncated form of it (N-EoCen), which were previously constructed by our group, were expressed and purified. The final product was examined by SDS-PAGE, and all of the protein products were of high purity.Using the fluorescence spectra, electrophoresis, electrochemistry methods and so on, we characterized the metal ions (Ca2+/Tb3+) binding properties of N-EoCen and its mutants. The experiments of aromatic residue-sensitized Tb3+ energy transfer and resonance light scattering demonstrated that: N-D73K could bind two Tb3+, which was similar to N-EoCen (WT-EoCen bound four Tb3+), and N-D37K could only bind one Tb3+. Based on fluorescence titration curve of N-D37K, the conditional binding constants of the N-EoCen loopⅡwere quantitatively found to be KⅡ= (8.31±0.18)×104 M-1 and KⅡ=(0.94±0.12)×102M-1 with Tb3+ and Ca2+, respectively.Using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated N-D37K mutant. Meanwhile, the conditional binding constants of TNS with Tb3+ -loaded N-EoCen and N-D37K were obtained, K(Tb3+-N-D37K-TNS)=(6.57±0.89)×106M-1, K(Tb3+-N-EoCen-TNS)=(9.35±0.05)×105 M-1. Using resonance light scattering (RLS) experiment and the native polyacrylamide gel electrophoresis (PAGE), we investigated the metal ions dependent self-assembly of N-EoCen, N-D73K and N-D37K, and the results demonstrate that self-assembly behavior of N-D37K is much weaker compared with N-EoCen and N-D73K, and cross-linking experiment exhibit the same result. Other physical and chemical factors, including ionic strength and pH, further demonstrate the previous results.It could be concluded that when Asp37, the first amino acid of loop I, was mutated to lysine, the binding site I lost its ability of metal ion-binding, indicating that Asp37 plays an important role in the binding of metal to N-terminal domain of EoCen. The binding process of Eu3+ with N-EoCen has been investigated by cyclic voltammetry and electrochemical impedance spectroscopy. |
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