Studies On The Nature Of C-terminal Domain Of Centrin And Phosphorylation

Centrin is the centrosome resident protein, low molecular weight (Mr-2000) which closely related intracellular fiber contraction, cell division, and separation of the spindle, etc. The reversible phosphorylation of proteins ranks among the most important possttranslational modifications that occur in the cell and also plays an important role in signal transduction and cellular regulation in vivo. Thus, phosphorylation and dephosphorylation in the body of the behavior provides an important regulatory mechanism of intracellular signal transduction. The centrin molecule contains four domains, each domain can be combined with a metal ion. Wherein Ⅰ, Ⅱ binding sites in the protein N-terminal domain; Ⅲ, Ⅳ binding sites located at the C-terminal domain. This selection of centrin C-terminal half molecules for the study because we focusing on the centrin C-terminal half molecules before and after phosphorylation by protein kinase A-mediated with the nature of the interaction, between the metal ions and chemical cross-linking.Firstly, a recombinant plasmid pGEX-6p-C-EoCen be constructed by the use of molecular biology methods, then recombinant plasmids were transformed into E. coli BL21(DE3). Expressed engineered bacteria and the activation of a single colony was picked, culture induction, collected by centrifugation, ultrasound lysate by GST affinity chromatography, PPase digested protein concentrated treatment C-EoCen. SDS-PAGE analysis showed that the resulting protein does not contain the hybrid protein bands experimental desired purity is reached. Sm3+binding with C-EoCen results in the conformation of C-EoCen changed from closed state to open state and is accompanied by enhancement of the exposed hydrophobic surfaces. In addition, the α-helix content of C-EoCen is increased by virtue of addition of Sm3+. By introducing competitive ligand xylenol orange, the conditional binding constants of site III and IV on C-EoCen with Sm3+are calculated to be lgKⅢ=6.23±0.39and lgKⅣ=6.81±0.51, respectively.Secondly, Native-PAGE and TNS fluorescence experiments shows that C-EoCen can be phosphorylated by PKA. Hydrophobic area exposed by the phosphorylation of the C-EoCen reduces pre-phosphorylated protein. It can be found that phosphorylation of C-EoCen in the presence of metal ions Ca2+, Tb3+, Gd3+was inhibited.At last, the C-EoCen before and after the phosphorylated can be cross-linked by chemical glutaraldehyde.