Studies On The Characterization And Function Of Euplotes Octocarinatus Centrin

Many dynamic processes in cells are critically dependent on the microtubule-based cytoskeletons.The number,direction and polarity of microtubules in eukaryotic cells are usually organized by microtubule-organizing centre(MTOC).In higher eukaryotes,such as humans,the centrosome functions as the MTOC.The composition of MTOC is highly proteinaceous,and there exists significant structural heterogeneity between them in different eukaryotes.Despite this heterogeneity,MTOC in all eukaryotes contain a number of conserved proteins.Centrosome consists of two vertical centrioles and pericentriolar material(PCM).There exist numerous proteins and the centrin was found to locate in it. Centrin is an acidic,low-molecular weight protein and belongs to the calcium binding super-family.One centrin molecule contains four helix-loop-helix motifs called EF-hands, one pair of which is capable of binding two calcium ions(Ca²⁺).Like calmodulin(CAM), centrin is comprised of two globular domains connected by a central linker.By polymerase chain reaction(PCR) and recombinant DNA techniques,expression plasmids ofâ‘ pGEX-6p-N-EoCen,â‘¡pGEX-6p-SC-EoCen,â‘¢pGEX-6p-LC-EoCen,â‘£pGEX-6p-M-EoCen(G151W),⑤pGEX-6p-M-SC-EoCen(G151W),â‘¥pGEX-6p-M-LC-EoCen (G151W) were were constructed.Fusion expressiones in E.coil BL21 were performed by induction of IPTG.The fusion proteins of GST-centrins were digested by PPase and purified by GST chelating affinity chromatography,ion exchange chromatography and molecular sieve chromatography.The final products were analyzed by SDS-PAGE.Interactions between Euplotes octocarinatus centrin(EoCen) and Ca²⁺,between EoCen and Tb³⁺ were investigated by circular dichroism spectra and fluorescence spectra at neutral condition.Results suggested that Ca²⁺ and Tb³⁺ induced secondary and terdiurnal conformation chages of protein.With the binding of metal ions to protein, EoCen changed from closed sate to open state resulting in the exposure of larger hydrophobic surfaces.Ca²⁺ and Tb³⁺ shares same metal ions binding sites on EoCen. C-terminal domain was identified to be sites with high affinity.The four EF-hand loops of EoCen exhibits different affinities for Ca²⁺ and Tb³⁺ with the orderⅣ＞Ⅲ＞Ⅱ,â… . According to resonance energy transfer of Trp to Tb³⁺ and ions competition of Ca²⁺ with Tb³⁺,the affinities between Tb³⁺(Ca²⁺) andâ…£,â…¡,â… on EoCen were calculated.The binding of melittin with EoCen,N-EoCen,SC-EoCen,LC-EoCen,respectively, was investigated by circular dichroism spectra,non-denaturalization PAGE,UV-Vis spectra,fluorescence spectra.Results suggested that only EoCen,SC-EoCen interacted with melittin.By virtue of D-helix holding the peptide binding site,LC-EoCen can not bind further with melittin.SC-EoCen was recognized as the main binding site of protein. In a metal ions-dependent manner,melittin binds with EoCen at the ratio of 1:1 and with affinity of 10⁷ M^-1.With the binding of melttin with protein,peptide turns slowly and results in the enhancement of fluorescence polarization.Ca²⁺,Tb³⁺ binding with EoCen leads to the conformational changes of EoCen leading to exposure of larger hydrophobic areas.The trp on melittin was embedded in the hydrophobic pockets,which was formed by the hydrophobic residues on EoCen.Different from calomdulin,EoCen interacts with mimic target peptide through its C-terminal domain.All of experiments including resonance light scattering(RLS),pull-down, non-denaturalization PAGE,yeast two-hybrids suggested that EoCen aggregated into polymer in vivo and in vitro.Metal ions play important role during the EoCen polymerization.The conformation induced by metal ions may be favorable for protein polymerization.Apart from the extended～20 residues in the N-terminal domain, C-terminal of EoCen also mediates polymerization of protein.After eliminating the extended residues by biological method,EoCen all the same polymerizes.Together into consideration,EoCen formed polymer in a mode of N-to-N(between N- and N-terminal domains) and C-to-C(between C- and C-terminal domains).Maybe,electronic static and hydrophobic forces droved this processes.Mg²⁺ is antagonistic to the interactions between metal ions Ca²⁺,Tb³⁺ and EoCen, between melittin and EoCen.Mg²⁺ binds maybe to EoCen at the same site with Ca²⁺ and the only difference exists in the different ligands bound to them.Ca²⁺ and Tb³⁺ binding to EoCen changed the conformation of protein from dumbbell into spherical state,resulting in the exposure of hydrophobic areas on EoCen.The presence of Mg²⁺ weakened this effect at neutral pH.And the Trp was embedded in the hydrophobic cavum.In the presence millimolar concentration of Mg²⁺,melittin binds with EoCen at the ratio of 1:1. The Mg²⁺ decreases the affinity between EoCen and melittin.In addition,lutecium promotes growth coliform containing pGEX-6p-EoCen.