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Abstract: Centrin is a low molecular mass (20KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb3+-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb2-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV–vis spectra and the fluorescence spectra methods. In 100mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb2-C-EoCen, four new peaks were observed at 265nm, 278nm, 317nm and 360nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480nm to 445nm in the presence of Tb2-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb2-C-EoCen were calculated to be log K(C-EoCen-TNS)=5.32±0.04 M−1 and log K(Tb2-C-EoCen-TNS)=5.58±0.12M−1, respectively. In addition, the protein of Tb2-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb2-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka′=6.79±0.17M−1. [Copyright &y& Elsevier]

Abstract: Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins, which often associated with the centrosomes and basal bodies. To explore the possible structural role of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu3+. The dependence of Lu3+ induced self-assembly of EoCen on various chemical and physical factors, including temperature, protein concentration, ionic strength and pH, was characterized using resonance light scattering (RLS). Control experiments with different metal ions suggest that Ca2+ and Lu3+ bindings to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu3+ exhibits the stronger effect, however, Mg2+ alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength demonstrate that the lutetium(III)-dependent self-assembly is closely related to the exposure of hydrophobic cavity. Control experiment on pH value with EoCen and the fragments of it, N-terminal domain of EoCen (N-EoCen), indicates that the electrostatic effect is of small tendency to be served as the main driving force in the self-assembly of EoCen. The specific oligomerization form of the protein was exhibited by cross-linking experiment. [Copyright &y& Elsevier]

Abstract: Centrin is a low molecular mass (20kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Centrin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity. To understand how centrin function, the glycine in position 115, the sixth amino acid of the loop of the protein''s third EF-hand, was converted into tryptophan (Trp). Local and overall changes were monitored for interactions between cations and Euplotes centrin and the mutant using fluorescence and UV spectroscopies. The results show that the binding of four Ca2+ ions to centrin with two higher affinity (sites III and IV) and two lower affinity (sites I and II), and the relative affinity of site IV is higher than that of site III. The secondary structure change of centrin and the mutant resulting from metal ions binding was measured by CD spectra. Centrin undergo a conformation change induced by Ca2+ binding from “closed” to “open”, through 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe, the different contributions to conformation change from the different sites were investigated, providing some insights into the molecular mechanism of centrin functions in the cell. [Copyright &y& Elsevier]