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

Abstract

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.

Structural Dynamics of RNA in the Presence of Choline Amino Acid Based Ionic Liquid: A Spectroscopic and Computational Outlook

“… 18 , 24 − 27 Furthermore, they are suitable for long-term storage and extraction of macromolecules. 15 , 28 − 30 The suitability of ILs for storing proteins and DNA is widely practiced since these macromolecules can retain their functionalities. 31 − 36 However, in the case of RNA, their high vulnerability to degradation with external factors like temperature, pH, and enzymes pose a difficult task to store them for an extended period.…”

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

“…In general terms, it could be a helpful approach to increase the stability of dried proteins (e.g., lyophilized form) [ 38 ] or proteins immobilized in solid matrixes [ 39 ]. Other works from the literature present alternative strategies to improve long-term stability of Cyt-c, such as storage in an aqueous solution of cholinium-based ionic liquids [ 40 ], aminoacid-based ionic liquids [ 41 ], surface active ionic liquids [ 42 , 43 ] and deep eutectic solvents (e.g., choline chloride ([Ch]Cl) and ethylammonium chloride (EAC)) [ 44 ]. However, these alternative solvents, such as deep eutectic solvents and ionic liquids, are not yet approved in pharmaceutical formulations.…”

Efficient Extraction of the RuBisCO Enzyme from Spinach Leaves Using Aqueous Solutions of Biocompatible Ionic Liquids

“…Most of the time, imidazolium-based ILs are not the best option in the field of SLE of biomolecules when considering their cost, toxicity and low biodegradability [20,[24][25][26][27]. Aiming at moving towards more sustainable solvents, cholinium-based ILs and analogues of glycine-betaine-based ILs (AGB-ILs) can be seen as promising alternatives in the field of extraction of proteins from biomass [24,28]. If properly selected, cholinium-based ILs preserve the tertiary structure of proteins and have lower toxicity than imidazolium-based counterparts [28].…”

“…Aiming at moving towards more sustainable solvents, cholinium-based ILs and analogues of glycine-betaine-based ILs (AGB-ILs) can be seen as promising alternatives in the field of extraction of proteins from biomass [24,28]. If properly selected, cholinium-based ILs preserve the tertiary structure of proteins and have lower toxicity than imidazolium-based counterparts [28]. On the other hand, AGB-ILs also display low cytotoxicity and ecotoxicity, supporting their direct use in nutraceutical extracts envisioning human consumption [24,29].…”