The Aggregation Properties Analysis Of Human Centrin 3

Centrin is a kind of acid calcium binding protein with relatively small molecular weight(about 20 k D).It is part of the EF-hand superfamily protein,closely related to calmodulin(Ca M),and has obvious calcium binding characteristics.Originally,centrin was found in unicellular green algae as part of the contractile fibers associated with the basal body.Four human centrin1-4(abbreviated Hscen1-Hscen4),there are differences in subcellular localization and biological function.Centrin has many functions.In cells,centrin is took part in signal transduction through nucleotide excision and repair,photoreceptor cell light transduction cascade,nuclear m RNA export mechanism and so on.Centrin plays an important role in centromeric localization,orientation,mitosis and in Spindle Pole Body(SPB)separation and microtubule tissue functions.And it is closely related to fiber contraction and cell division in cells.Abnormal centrin function is closely associated with human disease.Based on the previous studies on the properties of HsCen1 and Hscen2,this paper discussed the related properties of HsCen3widely expressed in somatic cells:Firstly,Tb³⁺sensitized fluorescence and isothermal titration calorimetry were demonstrated that HsCen3 can bind four Tb³⁺and the order of four metal binding sites was:?,?>?>?.It was proved by isothermal titration calorimetry that the four metal ion binding constants of HsCen3 and Tb³⁺were(5.9±1.5)×10⁵M^-1,(15.0±4.2)×10⁵M^-1,(52.2±1.3)×10⁵M^-1,(0.3±0.1)×10⁵M^-1.The effect of metal ions on the secondary structure of centrin was investigated by circular dichroism.Results suggested that metal ions change the secondary structure and increase the?-helix content of HsCen3.Conformational change of HsCen3 binding metal ions was detected by TNS(2-p-toluidinylnaphthalene-6-sulfonate).The results indicated that HsCen3 occur conformational change induced by metal ions,with hydrophobic surface exposed.HsCen3 has larger conformational change induced by Tb³⁺than Ca²⁺.Then,the thermodynamics of Lanthanide(Ln³⁺)and Ca²⁺-induced HsCen3 aggregation,as well as the effects of HsCen3 concentration,ion strength,ionic radius,TNS and p H on protein aggregation were studied by fluorescence resonance light scattering.The results indicated that Ln³⁺and Ca²⁺could induce HsCen3 to form oligomer.The effects of Ln³⁺on HsCen3aggregation were significantly more obvious than that of Ca²⁺.The N-terminal domain plays a key role in the formation of HsCen3 aggregates.It is speculated that electrostatic force and hydrophobic interaction are the main driving forces for HsCen3 aggregation.Finally,the binding of C-HsCen3 to mimic melittin was studied.Mimic melittin binds with C-HsCen3 at the ratio of 1:1 and the binding were independent of Ca²⁺Interactions between C-HsCen3 and melittin were investigated by circular dichroism.After binding with melittin,the?-helix content of protein increased and secondary structure changed.