Thermal stability and refolding of Hen Egg-White Lysozyme in aqueous Deep Eutectic Solvent solutions

Deep Eutectic Solvents (DESs) are mixtures that consist of various combinations of anions and cations derived from Lewis and Bronsted acids and bases. DESs are regarded as environmentally friendly and sustainable alternatives to ionic liquids, mainly due to their desirable characteristics such as low volatility, low cost, biodegradability, and straightforward synthesis. Experimental investigations have demonstrated that DESs and their aqueous solutions can contribute to preserving protein structure, enhancing thermal stability, and even facilitating partial refolding. However, these experiments have certain limitations, and the interactions between the solvents and proteins remain unclear. Therefore, we conducted molecular dynamics (MD) simulations using the native structure of Hen Egg-White Lysozyme (HEWL) solvated in a DES known as Reline, composed of choline chloride and urea, as well as its aqueous solutions. In particular, the simulations were designed to study three key aspects: (i) the stability of the HEWL structure at room temperature, (ii) the stability of the HEWL at a high temperature when the unfolding occurs, and (iii) the refolding of the thermally unfolded HEWL structure in Reline and aqueous reline solutions at room temperature. The results showed that at room temperature the most effective preservation of the HEWL native structure was observed in reline and the least diluted reline solution with 10% wt. water with a reduced number of H-bonds between HEWL and urea, possibly due to the high viscosity of the DES. Similarly, the thermal stability of the HEWL was observed in the systems with reline and the least diluted reline solution. Moreover, the most effective refolding of the HEWL was observed in the presence of reline (with 63% of complete refolding) and in the system with the least diluted reline solution (with 50% of complete refolding). Overall, the results of the MD study revealed promising applications of DESs and their aqueous solutions for protein thermal stability with possible refolding of the unfolded structures at room temperature.