Studies On The Properties And Functions Of Euplotes Octocarinatus Centrin

Centrin (caltractin) is a member of the calmodulin (CAM) superfamily ofEF-hand calcium-binding proteins. These proteins contain fourhelix-loop-helix domains, the so-called EF-hands, which may each bind oneCa²⁺. It is an essential component of the centrosomal structures (basal bodyor spindle pole body) in a wide range of organisms, ranging from algal andyeast and protozoa to higher plants and humans. Centrin is required forproper cell division, and plays important roles in contraction of centrin-basedfiber systems in eukaryotic cells as well.Euplotes octocarinatus is a unicellular protozoa, locate in a specialphylogenic degree showing a number of exceptional features. In present study,we investigated the characteristic and function of Euplotes octocarinatuscentrin, 20 KD, including 168 amino acids, no cysteine and tryptophan. Thepresent study, on the one hand, can help to clarify the many properties oflower eukaryotes, such as cell division, mobile cell; on the other hand, shedmore light on chemical bases of rare earth effect using centrin as possiblebinding target model.In order to further understand the function of centrin, 4 mutant gene(G115W,D37K,D73K,D146K) and 2 fragments gene (P23,P123) wereobtained using PCR technology. The sequence of the clones were confirmedby commerical company. The clones were digested by enzyme and ligatedinto expression vector pGEX-6p-1. Recombinant plasmids were transformedin E.coli BL21 (DE3) and induced by IPTG, thus soluble fusion protein wereexpressed. The proteins were purified to above 95% of purity by GST affinitychromatography and ion exchange chromatography Hitrap Q.Firstly, using the UV different spectra, CD spectra, and fluorescencespectra, we characterized the metal ions (Ca²⁺/Tb³⁺) binding property of Cen.The results showed that binding of metal ions (Ca²⁺/Tb³⁺) to protein changedthe secondary structure of Cen, the content ofαhelix was increased, andchanged the chemical environment of Tyr amino acid. Compared the properties of Cen induced by Ca²⁺ with Tb³⁺, range from CD spectra toaggregation characterization and conformation change measured by TNS, thefollowing conclusion can be drawn that Tb³⁺ and Ca²⁺ has very similarproperty in the binding to Cen. So we used the Tb³⁺ as probe to study Ca²⁺property in this study. Through Tb³⁺ sensitized fluorescence to study wildtype of Cen and G115W, the results showed that siteâ… andâ…¡of N-terminalare lower affinity sites, the siteâ…£and siteâ…¢of C-terminal are higheraffinity sites, and siteâ…£has higher affinity than siteâ…¢. Compared the Tb³⁺sensitized fluorescence curve of Cen with D37K,D73K and D146K,indicated that amino acid D exist respectively in first position of four metalbinding sites play important role in the binding of metal to protein. P23 andP123 have two sites and three sites, respectively.The aggregation characterization of Cen may be responsible for its fibercontact function, which was examined by resonance light scattering andnature PAGE in vitro. The results showed that self-assembly of Cen is metaldependent, which can be divided into two phase, the extent of aggregation ofN-terminal induced by metal ions is larger than C-terminal. Theself-assembly of D37K,D73K,D146K is impaired owing to failing to bindmetal ions in siteâ… ,â…¡andâ…£respectively. The self-assembly extent of Cen,P123 and P23 is gradually reduced, suggesting that each sites take part inself-assembly. The aggregation of P23 is severely weakened owing to lack ofN-terminal, indicating N-terminal may play more important role in thisprocess. Nature-PAGE experiment proved also this point. The self-assemblyof Cen was decreased with the increase of ion strength or TNS concentration,which showed that hydrophobic and electrostatic interaction are togetherresponsible for polymerization. Besides, the self assembly of Cen isinfluenced by the protein concentration, temperature and pH. Tb³⁺ has largereffect than Ca²⁺ in the process of aggregation.Conformational change of centrin was monitored by TNS. The resultsindicated that centrin occur conformational change induced by metal ions,with hydrophobic surface exposed. N-termianl has larger conformational change than C-terminal when bound to metal ions. The process can also bedivided into two phase, which is corresponding to self-assembly of centrin.Melittin was often used as model peptide in the study of interaction ofcalmodulin and target enzyme/protein. In present study, using the melittin asmodel peptide, the interaction of melittin and Cen, the mutantprotein(G115W) and fragment (P23, P123) were investigated. The resultsshowed that Cen can bind melittin by the molar ration 1:1 in the absence ofmetal ion in 10 mM Hepes, pH 7.4; C-terminal is responsible for binding ofmelittin, N-terminal did not bind to melittin. Suggesting that C-terminal andN-terminal plays different biological function through comparing theself-assembly and melittin binding experiment.Finally, the interaction of Cen and trifluoperazine(a CaM antagonist)was monitored by fluorescence spectra. We found that one Cen molecule canbind four trifluoperazine, the binding of trifluoperazine to Cen can decreasethe self-assembly of Cen, impair significantly binding of mellitin to Cen,indicating that trifluoperazine may be Cen antagonist.