Elucidating the Role of Thermal Flexibility of Hydrogels in Protein Refolding.

Abstract

Synthetic chaperones are stimuli-responsive materials that facilitate the refolding of denatured proteins in their native form and release refolded proteins in solution in the presence of external stimuli. Thermoresponsive hydrogels are a class of synthetic chaperones that require heat as an external stimulus to refold the denatured proteins into their biologically active conformations; however, the mechanism by which these hydrogels participate in protein refolding mechanism remains unclear. In this study, we explore the role of physical and structural changes in the hydrogel matrix that may participate in protein refolding efficacies of thermally responsive poly(vitamin B5 analogous methacrylamide) poly(B5AMA) hydrogels. Poly(B5AMA) hydrogels of different net charges, hydrophobicity, and cross-linking densities are synthesized by radical polymerization method and are evaluated for the restoration of enzymatic activity of thermally denatured enzymes (lysozyme and carbonic anhydrase) as a function of temperature and time and for the presence of residual water in the hydrogel architecture. The hydrogels with promising protein renaturation efficacies are further evaluated for their interactions with denatured proteins, and the role of thermal flexibility of the hydrogel matrix in protein refolding capabilities is elucidated.