Effects Of Crowding Systems On The Refolding And Unfolding Of Recombinant Human Brain-type Creatine Kinase

In vitro, folding of proteins has been extensively investigated, usually in dilute solution systems with low protein concentrations. However, this environment differs from that encountered within living cells, which contain high concentrations of various soluble and insoluble macromolecules, including protein, nucleic acids, ribosomes, carbohydrates and so on. The intracellular environment, described as "macromolecular crowding", has a significant effect on the folding of protein. Creantine kinase is an attractive model for studying protein folding. However, there is little report on the folding of human brain-type creatine kinase, especially for thoses in crowding systems. In the present study, we firstly quantitively investigated the effects of macromolecular crowding system on the folding of recombinant human brain-type creatine kinase (rHBCK), then the effects of the mixed crowdng systems including macromolecular crowding agent and osmolytes were also examined with a comparative method. The enzymatic activity, conformation and aggregation of rHBCK folding were examined with equilibrium and kinetics analysis. The molecular docking simulations of osmolytes and denaturant with rHBCK were also conducted. From all of the results, mechanisms of rHBCK folding in crowding systems were proposed. The results of the study were as follows:(1) It was found that PEG2000and dextran70could prevent rHBCK against the denaturation induced by0.5M guanidine hydrochloride (GdnHCl). The protective effects of200g/L PEG2000on the enzymatic activity and conformation of rHBCK were the best of all, with35%activity remained after1h denaturation, altering the kinetics of rHBCK unfolding from biphasic first order reation to monophasic first order reaction, giving a blue-shift of the maximum rHBCK’s peak from336.5nm to332nm; The protective effects of mixed macromolecular crowding on rHBCK unfolding were not better than those of single macromolecular crowding; There should be more intermediates when rHBCK unfolded in macromolecular crowding system, according to the results of phase diagram. It was clear that, based on the stablization of unfolding intermediates and decrease of unfolding rates, the macromolecular crowding could prevent rHBCK against denaturation.(2) Macromolecular crowding systems could promote the aggragation, and decrease the refolding yields and rates during the process of rHBCK refolding. The amount of aggregated protein was increased from13%to67%when100g/L PEG2000was added into the system. which was even more serious in mixed macromolecular crowding systems containing CT DNA and PEG2000(or dextran70); The refolding yields of rHBCK could be decreased from70%to20%,52%and57%when rHBCK unfolding in macromolecular crowding composed of100g/L PEG2000,100g/L dextran70and15g/L CT DNA, respectively. It is clear that, based on the promotion of aggregation and alteration of refolding rate, the macromolecular crowding could disturbe the refolding of rHBCK.(3) Mixed crowding systems containing osmolytes and macromolecular crowding agents could prevent rHBCK against the denaturation induced by0.8M GdnHCl, with an osmolyte-concentration dependent manner. Compared to those in the dilute solutions, osmolytes could decrease unfolding rates of fast and slow phases to more extent in macromolecular crowding systems, and showed better performances on the protection of the rHBCK’s conformation in those systems; The results of molecular docking simulations of osmolytes and denaturant with rHBCK indicated that the deactivation of guanidine may have been blocked by osmolytes because they share common binding sites in rHBCK. It is clear that, based on stablization of unfolding intermediates and native rHBCK state and decrease of unfolding rates, osmolytes could prevent rHBCK against denaturation in macromolecular crowding systems.(4) Osmolytes could increase the refolding yields and supress the aggregation during the process of rHBCK refolding in macromolecular crowding systems. Comparing to those in dilute solutions, the rcolding yields and rates of rHBCK could be increased to more extent by the presence of osmolytes in macromolecular crowding systems, in which the refolding yield could be increased from19%to49%by the presence of150g/L glycecrol; Aggregation of rHBCK refolding was inhibited by the presence of sucrose, glycerol and mannitol, respectively, with the amount of aggregation was decreased from68%to18%by the presence of400g/L sucrose in macromolecular crowding systems. It is clear that, based on stablization of refolding intermediates, accelerating the refolding rates, and inhibition of aggregation, osmolytes could help rHBCK refold in macromolecular crowding systms.In this study, differences of rHBCK folding in dilute solutions and macromolecular crowding systems were found when we examine the effects of crowding systems on the rHBCK folding. The goals of the investigation were to simulate an intracellular envrionment and develop the mixed crowding notion, to reinforce the growing appreciation for the need to understand the protein folding in vitro cellular process.