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.

Elucidating the Role of Optical Activity of Polymers in Protein–Polymer Interactions

“…Messina et al demonstrated that the stereoisomers of poly(trehalose) show limited interactions and no stabilization effect of proteins at a polymer/protein w / w ratio of 1, whereas an enhanced stabilization effect was observed at a polymer/protein w / w ratio of 10 under mechanical stress [ 6 ]. We have also previously demonstrated that poly(B5AMA)-based hydrogels act as macromolecular chaperones and stabilize the tertiary structure of LYZ at elevated temperatures [ 31 ]. The chaperone-like activities of these hydrogels were attributed to the hydration capability of hydrogels as a function of temperature providing protein stabilization via hydrogen bonding and hydrophobic interactions [ 31 ].…”

“…We have also previously demonstrated that poly(B5AMA)-based hydrogels act as macromolecular chaperones and stabilize the tertiary structure of LYZ at elevated temperatures [ 31 ]. The chaperone-like activities of these hydrogels were attributed to the hydration capability of hydrogels as a function of temperature providing protein stabilization via hydrogen bonding and hydrophobic interactions [ 31 ]. The enhanced thermal stability of BSA in the presence of stereoisomers of poly(B5AMA) further validates that the hydration potential of these polymers is the key factor in protein stability.…”

“…We have recently reported the synthesis of biomimetic polymers of vitamin B5 analogous methacrylamide (B5AMA) through a living radical polymerization approach and have shown that the poly(B5AMA) produced is highly hydrophilic and exhibits antifouling properties when grafted on a solid support [ 28 , 29 , 30 , 31 ]. Owing to the strong hydration tendency, poly(B5AMA) of different molecular weights exhibited the depletion aggregation of bacteria [ 29 ], and surface-tethered poly(B5AMA) chains showed low fouling behaviour for both proteins and bacteria [ 28 ].…”

Green Hydrogel Synthesis: Emphasis on Proteomics and Polymer Particle-Protein Interaction

“…The results indicated that protein–hydrogel surface interactions, rather than the degree of protein confinement, were most relevant for protein behavior within the gel, since the stabilization of protein structure was observed at both 4 and 10% crosslinking [ 123 ]. Kabir et al studied the enzyme refolding efficiency of hydrogels synthesized from vitamin B5 analogous methacrylamide (B5AMA), which function as thermoresponsive synthetic chaperones [ 124 ]. The results suggested that the hydrogen bonding interactions between the polymer and residual water were important for hydrogel thermal flexibility, which promotes enzyme refolding.…”