Exploring the role of hydrophilic amino acids in unfolding of protein in aqueous ethanol solution.

Hydrophobic association is the key contributor behind the formation of well packed core of a protein which is often believed to be an important step for folding from an unfolded chain to its compact functional form. While most of the protein folding/unfolding studies have evaluated the changes in the hydrophobic interactions during chemical denaturation, the role of hydrophilic amino acids in such processes are not discussed in detail. Here we report the role of the hydrophilic amino acids behind ethanol induced unfolding of protein. Using free energy simulations, we show that chicken villin head piece (HP-36) protein unfolds gradually in presence of water-ethanol binary mixture with increasing composition of ethanol. However, upon mutation of hydrophilic amino acids by glycine while keeping the hydrophobic amino acids intact, the compact state of the protein is found to be stable at all compositions with gradual flattening of the free energy landscape upon increasing compositions. The local environment around the protein in terms of ethanol/water number significantly differs in wild type protein compared to the mutated protein. The calculated Wyman-Tanford preferential binding coefficient of ethanol for wild type protein reveals that a greater number of cosolutes (here ethanol) bind to the unfolded state compared to its folded state. However, no significant increase in binding coefficient of ethanol at the unfolded state is found for mutated protein. Local-bulk partition coefficient calculation also suggests similar scenarios. Our results reveal that the weakening of hydrophobic interactions in aqueous ethanol solution along with larger preferential binding of ethanol to the unfolded state mediated by hydrophilic amino acids combinedly helps unfolding of protein in aqueous ethanol solution.