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摘要
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 microenvironment around Trp residue, the emission maximum of apoG115 W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCI. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCI. The unfolding of G115W induced by GdnHCI 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 ⊿〈△Gtotal0(H2O)〉 was used to determine the effect of Tb(Ⅲ) on the stability of apoprotein. The 〈△Gtotal0(H2 O)〉for Tb2-G115 W has a less increase of0.68 kJ/mol compared with apoG115W, proving Tb(Ⅲ) situated at C-terminal has negligible impact on the stability of protein. Whereas the <△Gtotal0(H2 O)> for Tb4-G115W has a rise of 1.29 kJ/mol compared with Tb2-G115W, manifesting Tb(Ⅲ) lcocated at low affinity sites has considerable influence on protein stability. mainly stabilizing the N-terminal domain.
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