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）.Intrinsi...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 ⊿〈△G_（total）~0（H_2O）〉 was used to determine the effect of Tb（Ⅲ） on the stability of apoprotein. The 〈△G_（total）~0（H_2 O）〉for Tb_2-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 〈△G_（total）~0（H_2 O）〉 for Tb_4-G115W has a rise of 1.29 kJ/mol compared with Tb_2-G115W, manifesting Tb（Ⅲ） lcocated at low affinity sites has considerable influence on protein stability. mainly stabilizing the N-terminal domain.展开更多
Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release...Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation, In the present study, the C-terminal of Euplotes octocarinatus eRF3 （eRF3C） and truncate eRF3C lacking 76 amino acids in C-terminal （eRF3Ct） were expressed in Escherichia coll. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purifled by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRFla in Euplotes octocarinatus.展开更多
基金Project supported by the National Natural Science Foundation of China(21571117)the PhD Programs Foundation of the Ministry of Education of China(20131401110011)
文摘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 ⊿〈△G_（total）~0（H_2O）〉 was used to determine the effect of Tb（Ⅲ） on the stability of apoprotein. The 〈△G_（total）~0（H_2 O）〉for Tb_2-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 〈△G_（total）~0（H_2 O）〉 for Tb_4-G115W has a rise of 1.29 kJ/mol compared with Tb_2-G115W, manifesting Tb（Ⅲ） lcocated at low affinity sites has considerable influence on protein stability. mainly stabilizing the N-terminal domain.
基金This work was supported by grants from National Natural Science Foundation of China(No.30670282,30470239,3030038)Natural Science Foundation of Shanxi Province(No.20041079,20051065).
文摘Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation, In the present study, the C-terminal of Euplotes octocarinatus eRF3 （eRF3C） and truncate eRF3C lacking 76 amino acids in C-terminal （eRF3Ct） were expressed in Escherichia coll. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purifled by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRFla in Euplotes octocarinatus.
