Disparity in stabilization effect of polyols on the cold- and heat-induced unfolding of apo-myoglobin: Impact of alkyl chain length and number of hydroxyl groups.

The fundamental challenge in protein-based therapeutics and biotechnological applications is maintaining protein stability and activity. This study investigates the effects of various diols (ethylene glycol, 1,2-butanediol, 1,4-butanediol, 1,2-hexanediol, 1,6-hexanediol) and triols (glycerol, 1,2,6-hexanetriol) on the cold and heat-induced denaturation of apo-myoglobin (apoMb). The polyols destabilize the protein during heat denaturation but stabilize it against cold denaturation, unlike conventional denaturants or stabilizers. These polyol-induced effects are enthalpically driven. Fluorescence studies indicate that longer alkyl-chain polyols induce early heat denaturation of the protein but maintain its compactness at cold temperatures. Replica-exchange molecular dynamics simulations highlight differences in protein-water and protein-polyol interactions between cold- and heat-induced unfolded states. At low temperatures, the water density around the protein is maintained in the presence of the polyols, thereby ensuring its stability. The effect is more pronounced in the presence of polyols with longer alkyl chains. Conversely, during the heat, preferential binding of longer alkyl-chain polyols to the protein surface disrupts the water network and non-bonded interactions, thus destabilizing the protein. Interaction energy analyses suggest that longer alkyl-chain polyols predominantly interact via van der Waals interaction at high temperatures, whereas the interaction is reduced at low temperatures. This study emphasizes that osmolytes can exhibit different interactions with proteins during cold and heat denaturation, in which the hydrophobic effect can play a prominent role.