Studies On The Mechanism Of Refolding And Aggregation Of Creatine Kinase

The rabbit brain type creatine kinase (RBCK), the human brain type creatine kinase (HBCK) and the rabbit muscle type creatine kinase (RMCK) were taken as model proteins to study the mechanism of the protein folding and aggregation.The effects of zinc ions on the activities of both RBCK and HBCK were studied. The results showed that the low concentration of zinc ions will inhibit the activity of the enzymes. The high concentration of zinc ions will induce the enzymes to aggregate. The aggregations of the enzymes depend on the temperature, the enzyme concentration and the zinc ion concentration. The effect of glycerol, sucrose, glycine, and proline on the aggregation of RBCK and HBCK induced by the zinc ions has been examined. The results showed that glycine and proline can effectively prevent aggregation of the enzymes, and fully recover the conformation and activity of the enzymes.We studied the cysteine modification of RMCK and measured the kinetic aggregation process of the enzyme. The results showed that the enzyme aggregation was influenced by pHvalue in the presence of zinc ions. We also found that cysteine modification at the active site with using 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) induced conspicuous aggregation in a dose dependent manner. This aggregation process was directly related with decreasing the transition free-energy (ΔΔGAG). DTT, glycine and proline were able to successfully block RBCK aggregation by increasing theΔΔGAG as well as by suppressing the hydrophobic RBCK surface, and to largely recover the enzyme activity which was inhibited by the zinc ions and DTNB. Our study suggested that the cysteine modification of the RMCK active site might play a key role for inducing unfavorable aggregation in a proper condition, and the aggregation followed first-order kinetics with the accompanying changes of transitional free-energy and the hydrophobic surface, and the method of the blocking aggregation was designed.The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications, in the presence of denaturant, at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Our study demonstrated monomer CK aggregation; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin mechanism, and these pathways followed first-order kinetics.Using bovine retinal creatine kinase chain A (PDB entry:1G0W) as a structural model, we predict the 3D structure of RBCK and HBCK with the homology modeling. We carry about the In Silico Docking experiments between zinc ions and the brain type creatine kinase, between RBCK and acrylamide, also between HBCK and SDS. The results showed: the zinc ions, SDS and acrylamide may combine with the active sites of the enzymes; the ARG132,ARG320 and ARG341 in HBCK may directly react with SDS, but the CYS74 and GLY73 in RBCK may directly react with acrylamide.Through the above results, we developed and polished the mechanism of the folding and aggregation of creatine kinase, predicted the combinant sites in sterology and explored the relationship between the folding and aggregation of creatine kinase and the neurodegenerative diseases.