Ya-Qin Zhao

Investigation on the Inclusion Behavior of ApoCopC with Vitamin B6

In neutral phosphate buffer solution of pH 7.0, the interaction between apoCopC and Vitamin B6 has been investigated in detail by means of fluorescence spectroscopy. According to the change of Vitamin B6 fluorescence spectra and fluorescence polarization, we can conclude that a novel supramolecular system is generated. ApoCopC can form a 1:5 host-guest inclusion supramolecular complex with Vitamin B6, and the formation constant has been calculated to be (2.24 ± 0.08) × 104 M− 1. It suggests the strong inclusion ability of apoCopC to the guest molecules. In addition, the mechanism of the apoCopC protein fluorescence quenching by Vitamin B6 was also discussed. And based on the Stern-Volmer equation, the apparent quenching constant was estimated to be (2.75 ± 0.05) × 104 M− 1.

Characterization of self-assembly of Euplotes octocarinatus centrin

Centrin, an EF-hand calcium-binding protein with high homology to calmodulin (CaM), is an essential component for microtubule-organizing center (MTOC) in organisms ranging from algae and yeast to human. It plays an important structural role by contributing to the formation of Ca(2+)-sensitive contractile filaments and some super-molecular assemblies. Previous work suggests that the N-terminal domain of centrin especially its first 20-residue fragment, is required for the self-assembly of protein. Native polyacrylamide gel electrophoresis (native-PAGE), pull-down assay, fluorescence resonance light scattering (RLS) and yeast two-hybrid assay indicate that the C-terminal domain of Euplotes octocarinatus centrin (EoCen) also contributes to the centrin self-assembly besides its N-terminal domain in vivo and in vitro. On the basis of our results, a self-assembly mode of centrin, which is C-to-C as well as N-to-N (between C- and C-terminal domains as well as between N- and N-terminal domains), is put forward providing maybe some insights into the molecular mechanism of centrin functions in the cell.

Fluorescence study on the interaction between apoCopC and cupric

The interaction between apoCopC and cupric was investigated by fluorescence spectra, in phosphate (20 mmol/L) buffer at pH 6.0. Results suggest that the environment is measured to be hydrophobic completely around tryptophan (83). At the same time, apoCopC fluorescence at 320 nm was significantly quenched with the addition of cupric and the 1:1 stoichiometric ratio of apoCopC to cupric was confirmed by fluorescence. In addition, the conditional binding constants were calculated to be KCu_Copc = (1.8±0.58)× 1013 mol−1 L on the basis of the results of fluorescence titration curves. The apoCopC has the ability to bind specifically cupric ion.

Stability of proteins with multi-state unfolding behavior

A new model used to calculate the free energy change of protein unfolding is presented. In this model, proteins are considered to be composed of structural elements. The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method. If a protein consists of the same structural elements, its unfolding will displays a two-state process, and only the average structural element free energy change 〈ΔGelement0(H2O)〉 can be measured. If protein consists of completely different structural elements, its unfolding will show a multi-state behavior. When a protein consists of n structural elements its unfolding will shows a (n+1)-state behavior. A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration, not to the start of each transition. The measured ΔGprotein0(H2O) is the sum of the free energy change for each structural element. Using this new model, we can not only analyze the stability of various proteins with similar structure and similar molecular weight, which undergo multi-state unfolding processes, but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change 〈ΔGelement0(H2O)〉. Although this method cannot completely provide the exact free energy of proteins, it is better than current methods.

The interaction between lanthanide (III) and N-terminal domain of Euplotes octocarinatus centrin

Centrin, a member of calcium-binding proteins, is an essential component for microtubule-organizing center (MTOC). Lanthanide (Ln) ions can increase amounts, enhance stability and orderliness of microtubules which is an important component of cytoskeleton. To investigate the structural basis of the effect of Ln ions on orderliness of microtubules, we focused on the interactions between the isolated N-terminal domain of Euplotes centrin (N-EoCen) and Ln by some combined biophysical and biochemical methods. Our results suggest that Ln ions may bind to the canonical calcium binding sites on N-EoCen. Taking advantage of ligand competition, we first determined the metal-binding affinities of Nd(3+), Eu(3+), Gd(3+) and Tm(3+) with N-EoCen. Major changes of N-EoCen in secondary and tertiary structure are noted while Ln ions bind with N-EoCen through CD spectra and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) fluorescence. N-EoCen exists in the form of monomer and dimer in the presence of Ln ions. These results can provide some insights into the structural basis of how Ln ions achieve biological effect in cell through the centrin protein.

Analysis of Lanthanide-Induced Conformational Change of the C-Terminal Domain on Centrin

Centrin, an EF-hand calcium-binding protein with high homology to calmodulin (CaM), is an essential component of microtubule-organizing center (MTOC). Lanthanide (Ln) ions can improve the stability, increase the amount and enhance the orderliness of microtubules, which are components of cytoskeleton. In order to investigate the structural basis of Ln ions on enhancing orderliness of microtubules, we characterized the binding properties of Ln ions with the isolated C-terminal domain of the Euplotes centrin (C-EoCen). Results suggested that Ln ions may occupy the canonical Ca2+ binding sites on C-EoCen with middle affinity. Near- and far-UV CD spectra of C-EoCen displayed pronounced differences before and after additing Ln ions. The asymmetry of microenvironments of Phe on C-EoCen was changed. Using 2-p-toluidinylnaphthalene-6- sulfonate (TNS) as probe, Ln ions induced C-EoCen to undergo conformational changes from closed state to open state, resulting in exposing hydrophobic patches to external environments. Ln ions have more obvious effect on the conformation of centrin than Ca2+. The differences found in the interactions of centrin binding with Ln ions/Ca2+ maybe provide some insights for structural basis of centrin functions in vivo.

Fluorescence spectra study the perturbations of CopC native fold by 2-p-toluidinynaphthalene-6-sulfonate.

2-p-Toluidinynaphthalene-6-sulfonate (TNS) was discovered to perturb native fold of CopC protein and to induce loss of biological activity to some extent which was dependent on TNS concentration. Hydrophobic and electrostatic interactions were revealed to account for the perturbation by comparison with some analogy. TNS, with far low concentration of 10(-5) to 10(-4)M, is presented as a denaturant. So TNS should be deliberated in detecting macromolecular conformation change as single evidence at higher concentration.

The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin

Centrin is a member of the EF-hand superfamily that is phosphorylated during mitosis and is associated with alterations of contractile fibers. To obtain insight into the structural basis for the functional effects of phosphorylation, we found that the serine residue at position 166 of Euplotes octocarinatus centrin (EoCen) can be phosphorylated by protein kinase A (PKA) in the absence or presence of metal ions using 31P-NMR spectroscopy. Cations of Ca2+ and Tb3+ bound to EoCen resulted in an important structural transition from a closed to an open state. EoCen in both the closed and the open state can be phosphorylated by PKA. After phosphorylation, secondary and tertiary structural changes of EoCen, mainly on its C-terminal domain (C-EoCen), were noted through circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and 2-p-toluidinylnaphthalene-6-sulfonate fluorescence. After the protein was phosphorylated, the α-helix content and the extent of the exposed hydrophobic surface on EoCen were decreased. Phosphorylated EoCen has higher affinity for the peptide melittin than nonphosphorylated EoCen. In addition, binding of melittin with phosphorylated C-EoCen was enthalpy-driven.Graphical Abstract

Analysis of the role of Mg2+ 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.