Studies On The Phosphorylation And Characterization Of Euplotes Octocarinatus Centrin

The reversible phosphorylation of proteins ranks among the most important posttranslational modifications that occur in the cell. Posttranslational modification by phosphorylation is a ubiquitous regulatory mechanism in both eukaryotes and prokaryotes, which plays an important role in signal transduction and cellular regulation. In addition, Intracellular phosphorylation by protein kinases, triggered in response to extracellular signals, provides a mechanism for the cell to switch on or switch off many diverse processes.In this work, we selected the Ciliate Euplotes octocarinatus centrin (EoCen) as an experimental material for the study of PKA-mediated protein phosphorylation.Firstly, one recombinant engineering bacteria (pGEX-6p-1-N+1-EoCen) was constructed, recombinated, expressed and purificated by molecular biology methods. In addition, we also expressed and purificated the EoCen, C-EoCen and N-EoCen by the some method.31P-NMR and the native-PAGE experiments show that PKA can catalyze the phosphorylation of EoCen. And the rare earth ion Tb3+had no significant effect on the PKA-mediated EoCen phosphorylation, but low concentration of Ca2+can promote the reaction and high concentration of Ca can inhibit the reaction. In addition,31P-NMR proved that EoCen had only one PKA phosphorylation site which is the Ser-166(KQTS) in C-terminal.Native-PAGE, CD spectrum and TNS fluorescence titration show that the PKA-mediated phosphorylation can reduce the α-helix and exposed hydrophobic surface of EoCen. The hydrophobic surface of phosphorylated proteins is smaller than unphosphorylated proteins, and the hydrophobic surface of terbium-saturated phosphorylated proteins is bigger than calcium-saturated phosphorylated proteins. Finally, the native-PAGE, CD spectrum and fluorescence titration experiments showed that the capacity of phosphorylated C-EoCen (P-C-EoCen) binding with melittin is stronger than unphosphorylated C-EoCen (C-EoCen), but the binding ratio is still1:1. By calculating the constants of the binding of melittin to EoCen before and after phosphorylation, as well as studying their conformational changes, this article proved that the main acting force between EoCen and melittin is the electrostatic force. In addition, series of thermodynamic parameters of the binding of melittin to EoCen before and after phosphorylation indicated that the reaction is an exothermic reaction, as well as a spontaneous reaction.