Ciliate Euplotes Octocarinatus Centrin Research Articles

Crystal structure of the trimeric N-terminal domain of ciliate Euplotes octocarinatus centrin binding with calcium ions.

Centrin is a member of the EF-hand superfamily of calcium-binding proteins, a highly conserved eukaryotic protein that binds to Ca2+ . Its self-assembly plays a causative role in the fiber contraction that is associated with the cell division cycle and ciliogenesis. In this study, the crystal structure of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) was determined by using the selenomethionine single-wavelength anomalous dispersion method. The protein molecules formed homotrimers. Every protomer had two putative Ca2+ ion-binding sites I and II, protomer A, and C bound one Ca2+ ion, while protomer B bound two Ca2+ ions. A novel binding site III was observed and the Ca2+ ion was located at the center of the homotrimer. Several hydrogen bonds, electrostatic, and hydrophobic interactions between the protomers contributed to the formation of the oligomer. Structural studies provided insight into the foundation for centrin aggregation and the roles of calcium ions.

Effect of Tb(III) on the unfolding of ciliate Euplotes octocarinatus centrin induced by guanidine hydrochloride

To understand the unfolding of ciliate Euplotes octocarinatus centrin (EoCen), the glycine positioned at 115, the sixth residue of the loop of the protein's third EF-hand, was mutated into tryptophan (Trp). Intrinsic fluorescence and Tb(III) binding properties of wild type EoCen and G115W mutant were monitored by fluorescence spectra in 10 mmol/L Hepes. The emission maximum of EoCen was 306 nm and mutation had no impact on the Tb(III) binding properties. The properties of G115W were investigated by fluorescence, far-UV circular dichroism (CD) spectra and fluorescence decays in the absence or in the presence of 6 mol/L guanidine hydrochloride (GdnHCl). For the increase in polarity of micro-environment around Trp residue, the emission maximum of apoG115W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCl. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCl. The unfolding of G115W induced by GdnHCl was assessed by using the model of structural element. The unfolding of proteins is a sequential reaction, namely two-transition, three-state process. The first transition belongs to the unfolding of the C-terminal domain, and the second transition is assigned to the unfolding of the N-terminal domain. The Δ〈ΔGtotal0(H2O)〉 was used to determine the effect of Tb(III) on the stability of apoprotein. The 〈ΔGtotal0(H2O)〉 for Tb2-G115W has a less increase of 0.68 kJ/mol compared with apoG115W, proving Tb(III) situated at C-terminal has negligible impact on the stability of protein. Whereas the 〈ΔGtotal0(H2O)〉 for Tb4-G115W has a rise of 1.29 kJ/mol compared with Tb2-G115W, manifesting Tb(III) located at low affinity sites has considerable influence on protein stability, mainly stabilizing the N-terminal domain.

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca2+ and Tb3+) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37>Asp146>Asp110>Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

A comparative study on binding ability of three lanthanide ions with centrin using impedance method

Centrin, an EF-hand calcium-binding protein, is involved in the formation of the microtubule organizing center (MTOC). The interactions between Gd3+, Eu3+, La3+ and the N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) were investigated by electrochemical impedance spectroscopy (EIS) using potassium ferricyanide as a redox probe. Results show that an increase in the lanthanide ion (Ln3+) concentration resulted in a lower impedance magnitude in the Nyquist plots when the N-EoCen-modified glassy carbon electrode (N-EoCen-GC) was used. The titrations of N-EoCen with Ln3+ confirm the complexation reactivity of Ln3+ with N-EoCen and suggest that the Ln2–N-EoCen complex was formed. In contrast, the binding ability of Ca2+ to N-EoCen is far less than that of Ln3+. The binding constants of the metal ions to N-EoCen are in the order of Gd3+ ≈ Eu3+ > La3+ ≫ Ca2+. Compared with the resonance light scattering (RLS) method, the EIS method can not only distinguish the binding ability of different metal ions to N-EoCen, but can also distinguish clearly the different metal ion binding sites on N-EoCen.

Electrochemistry of Eu(III) Binding to N-terminal of Euplotes Octocarinatus Centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand super family of calcium-binding proteins, which is associated with the centrosomes. In this work, the interaction between N-terminal of EoCen (N-EoCen) and Eu3+ was investigated by cyclic voltammetry and UV-Vis spectroscopies. The result shows that a 2:1 of Eu3+ to N-EoCen complex (Eu2-N-EoCen) is formed. The electrochemical characteristics of Eu2-N-EoCen at a multi-wall carbon nanotube/Nafion composite film modified glassy carbon electrode (MWNT/Nafion/GCE) were then investigated in detail, from which a direct, quasi reversible electron transfer of Eu2-N-EoCen had been obtained. According to the Laviron diffusionless-controled equations, the electron transfer rate constant ks, was calculated to be 2.33 ± 0.14 s−1. The pH dependence of the formal potential of Eu2-N-EoCen suggests that protonation accompanies electron transfer between pH 5.5–7.4. The electrochemical redox and quantitative analysis of Eu2-N-EoCen are therefore of great significance not only studying the physiological processes of Eu2-N-EoCen in organisms but also the interaction mechanism with target protein.

Adsorption of Euplotes octocarinatus centrin on glassy carbon electrodes as substrates to study europium–protein interactions

The adsorption of N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) onto a glassy carbon (GC) electrode was studied by cyclic voltammetry and electrochemical impedance spectroscopy. The adsorption process obeys Langmuir isotherm adsorption equation. Based on the adsorption, direct immobilization of N-EoCen onto the glassy carbon surface was used for construction of an N-EoCen-modified GC electrode. Then the electrode was used to probe the binding mode of europium ions with N-EoCen. The results show that with the increasing concentration of europium ions, the redox peaks of probe increase gradually. Simultaneously, the peak separation decrease and peak currents of the redox reaction increase. In addition, two binding sites in N-EoCen show no-equiv signal of CV change: the signal is slightly changed by the binding of the first europium ions and largely did by the binding of the second europium ions. It can be attributed to the change of conformation or aggregation of protein after binding of europium ions. It offers a viable model for illustrating the interaction of lanthanides with EoCen. Through the titration curve the process of combination can be explored.

Analysis of the role of Mg 2+ on conformational change and target recognition by Ciliate Euplotes octocarinatus centrin

The binding of Mg 2+ with the Euplotes octocarinatus centrin (EoCen) and the effect of Mg 2+ on the binding of EoCen with the peptide melittin were examined by spectroscopic methods. In this study, it was found that Mg 2+ may bind with Ca 2+-binding sites, at least partly, on EoCen, which displays ∼10-fold weaker affinity than Ca 2+. In the presence of Mg 2+, Ca 2+-saturated EoCen undergoes significant conformational changes resulting in decreased exposure of hydrophobic surfaces on the protein. Additionally, excess Mg 2+ did not change the stoichiometry, but rather reduced the affinity of EoCen to melittin. The Mg 2+-dependent decrease in the affinities of EoCen to melittin is an intrinsic property of Mg 2+, rather than a nonspecific ionic effect. The inhibitory effect of Mg 2+ on the formation of complexes between EoCen and melittin may contribute to the specificity of EoCen in target activation in response to cellular Ca 2+ concentration fluctuations.

Metal ion-binding properties of wild-type and mutant D37K of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue. The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen. Aromatic-sensitized Tb3+ fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation. Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of the EoCen loop II were quantitatively found to be KII = (1.61 ± 0.04) × 105 L mol−1 and KII = (3.52 ± 0.08) × 102 L mol−1 with Tb3+ and Ca2+, respectively. Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.

Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb3+ as a fluorescence probe, that EoCen has four calcium-binding sites. The sensitized emission arises from nonradiative energy transfer between the three tyrosine residues (Tyr46, Tyr72, and Tyr79) of the N-terminal half and the bound Tb3+ ions. To determine the most critical of the three tyrosine residues for the process of fluorescence resonance energy transfer, six mutants of the N-terminal domain of EoCen, which contain one (N-Tyr46/N-Tyr72/N-Tyr79) or two (N-Y46F/N-Y72F/N-Y79F) tyrosine residues, were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was most affected, displaying a 50% reduction compared with wild-type N-EoCen. Among the tyrosines, Tyr79 is the shortest mean distance from the protein-bound Tb3+ (at sites I/II), as calculated via the Förster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr79, which exists in a hydrophobic environment, has the highest quantum yield and a relatively long average lifetime. The decay of Tyr79 is the least heterogeneous among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr79 and the oxygen from the side chains of the residue Asn39. Kinetic experiments on tyrosine and Tb3+ fluorescence demonstrate that tyrosine fluorescence quenching is largely due to the self-assembly of EoCen, and that the quenching degrees of the mutants differ. Resonance light scattering and crosslinking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.

Electrochemical study of the interaction between Eu3+ and ciliate Euplotes octocarinatus centrin

A new species was formed when protein P23 (one segment of ciliate Euplotes octocarinatus centrin) was added to a solution of Eu3+. The interaction between P23 and Eu3+ was investigated by cyclic voltammetry, pulse voltammetry and electrochemical impedance spectroscopy in 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer (pH 7.4) using a pyrolytic graphite electrode. The formal potential (Eo′) of Eu3+ shifted from −0.61 to −0.84 V (versus saturated calomel electrode) after P23 was added to the Eu3+ solution. The diffusion coefficient (D), the charge-transfer coefficient (α) and the electron transfer standard rate constant (ks) were obtained in the absence and the presence of P23. The affinity constant of Eu3+ and P23 was determined to be (1.89 ± 0.51) × 104 M−1. The electrochemical investigation of europium bound to the protein provided useful data for the studies of calcium-binding proteins.

Role of Asp37 in metal-binding and conformational change of ciliate Euplotes octocarinatus centrin

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.

Investigation on the binding of TNS to centrin, an EF-hand protein

The interaction between 2- p-toluidinylnaphthalene-6-sulfonate (TNS) and ciliate Euplotes Octocarinatus centrin (Cen) has been studied by fluorescence spectroscopy. The binding constants of TNS with Cen were measured at different temperature in the 0.01 M Hepes, pH 7.4. The binding process is exothermic and involves a positive entropy change. The negative value of enthalpy predominately contributes to the negative free energy of binding between TNS and Cen. The salt (KCl) increases the association constant of TNS and Cen. These results and resonance light scattering experiment suggest that the binding force between TNS and Cen is hydrophobic. The distance ( r) between TNS and tryptophan of mutant G115W, which sheds more insight into the binding of TNS to Cen, was determined as 4.85 nm based on Förster non-radiative energy transfer theory.

Lutetium(III)-dependent self-assembly study of ciliate Euplotes octocarinatus centrin

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 Lu 3+. The dependence of Lu 3+ 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 Ca 2+ and Lu 3+ bindings to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu 3+ exhibits the stronger effect, however, Mg 2+ 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.

Spectral study on the interaction of ciliate Euplotes octocarinatus centrin and metal ions

Centrin is a low molecular mass (20 kDa) 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 Ca 2+ 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 Ca 2+ 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.

Metal ions-induced conformational change of P 23 by using TNS as fluorescence probe

In 10 mM N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid (Hepes), pH 7.4, 25 °C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P 23) induced by metal ions were investigated using 2- p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P 23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb 3+ has more larger influence than Ca 2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca 2-loaded or Tb 2-loaded P 23 were obtained, K (Ca 2-P 23 + TNS) = (7.49 ± 0.88) × 10 5 mol −1 L, K (Tb 2-P 23 + TNS) = (8.24 ± 0.49) × 10 5 mol −1 L.