Amino-Acid-Based Ionic Liquids for the Improvement in Stability and Activity of Cytochrome c: A Combined Experimental...

The demand for long-term storage and stability of proteins has increased substantially in the pharmaceutical industries, yet the sensitivity of proteins toward the environment has become a cardinal task for researchers. To deal with this, we have selected a multifunctional enzyme Cytochrome-c (Cyt-c) involved in many chemical and biochemical reactions as model protein, which is very sensitive and loses structural integrity on exposure to the environment. The remarkable features of ionic liquids (ILs) have entitled them as alternatives to aqueous and organic solvents for solubility, storage, and surrogate reaction medium. Hence, we have adapted the biocompatible and nontoxic cation and anion based amino acid ILs (CAAAILs) as potential solvents for storage and stability of Cyt-c. Herein, we report the molecular insights and thermodynamics of interaction between CAAAILs and Cyt-c with the help of isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), UV-vis, CD, and fluorescence spectroscopy as well as molecular docking and molecular dynamics (MD) simulations. The structure and stability of Cyt-c remain unchanged in the presence of CAAAILs. Both electrostatic and hydrophobic interactions are accountable for the binding of CAAAILs in the region between terminal helices and the loop of Cyt-c through nonspecific multiple binding sites, which can be exploited for storage and stability of proteins and will be helpful in designing new biobased ILs for biochemical applications.