Effect of Tb(III) on the unfolding of ciliate Euplotes octocarinatus centrin induced by guanidine hydrochloride

Abstract

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 micro-environment around Trp residue, the emission maximum of apoG115W at 343 nm is shifted to 359 nm in 6 mol/L GdnHCl. Also the secondary structure is lost nearly and fluorescence lifetime decreases in 6 mol/L GdnHCl. The unfolding of G115W induced by GdnHCl 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 Δ〈ΔGtotal0(H2O)〉 was used to determine the effect of Tb(III) on the stability of apoprotein. The 〈ΔGtotal0(H2O)〉 for Tb2-G115W has a less increase of 0.68 kJ/mol compared with apoG115W, proving Tb(III) situated at C-terminal has negligible impact on the stability of protein. Whereas the 〈ΔGtotal0(H2O)〉 for Tb4-G115W has a rise of 1.29 kJ/mol compared with Tb2-G115W, manifesting Tb(III) located at low affinity sites has considerable influence on protein stability, mainly stabilizing the N-terminal domain.