Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase

Abstract

The thermal stabilization and refolding of horseradish peroxidase (HRP) upon heating were investigated using an artificial molecular chaperone consisting of cholesterol-bearing pullulan (CHP) nanogels. The CHP nanogels inhibited the aggregation of HRP under heating by complexation with the denatured HRP. The enzyme activity of HRP complexed with CHP nanogels was not detected. However, the enzyme activity recovered up to 80% of native HRP after the addition of cyclodextrin (CD) to the complex. The dissociation of CHP nanogels was induced by the formation of an inclusion complex of cholesterol groups of CHP with CD. The enzyme activity of HRP was only significantly recovered by the addition of β-CD or its derivatives. Natural molecular chaperones, such as GroEL/ES, trap, fold, and release the nonnative proteins by changing the hydrophobicity of the specific sites of the molecular chaperone that interact with the nonnative protein. The functional mechanism of the nanogel chaperon system is similar to that of natural molecular chaperones. The nanogel chaperone system is a useful tool to aid the refolding and thermal stabilization of unstable proteins for post-genome research, and in medical and biological applications.