Discrepant Effects of Hydrated and Neat Reline on the Conformational Stability of a Knotted Protein

Although knotted proteins are rare in number, their peculiar topology has long intrigued the scientific community. In this study, we have explored the conformational stability of a trefoil-knotted protein, YbeA, in reline (choline chloride:urea in a 1:2 ratio), a well-characterized deep eutectic solvent, using classical molecular dynamics simulation. Deep eutectic solvents (DESs) are explored as a reliable alternative to conventional solvents, effectively altering a protein’s structural stability and activity, either stabilizing its native state or disordering its conformation depending on the relevant interactions involved. Here, using pure and hydrated concentrations of reline, we observe the conflicting effect of the DES on the knotted protein’s stability. Our studies at room temperature and elevated temperatures show that in pure reline, the protein is conformationally stable and rigid. In contrast, the protein tends to lose its structural integrity in hydrated reline. The stable knotted topology also gets untied as the protein, solvated in hydrated reline, is exposed to an elevated temperature. Using Minimum Distance Distribution Functions and Kirkwood–Buff Integrals, we analyzed the solvation pattern of the DES constituents around the protein. We expect that this study will lead to more effective strategies in developing tailored solvent systems for comprehending the conformational behavior of knotted proteins.