Insight into The Impact of Ionic Liquids On the Structure, Stability, And Activity of Proteins

The protein stability in aqueous solutions is a commonly concerned issue in various biological fields, especially in the pharmaceutical field for development of therapeutic protein products. Proteins are functional/active in the folded or native state. They easily undergo conformational changes reversibly or irreversibly in response to environmental conditions. In general, the stability of proteins depends on temperature, pressure, and most importantly on the solvent properties. Different types of co-solvents (Sugars, polyhydric alcohols, etc.) were reported in the past, which serve the purpose of stabilization to some extent. In recent years, ionic liquids (ILs) have emerged as a new class of solvents which can strengthen the stability of proteins. Although ILs are commonly used for the stabilization of biomolecules, the biomolecular interactions causing the effect, stabilization or destabilization are still an active subject of considerable interest. In this thesis, several ionic liquids based on imidazolium, ammonium, and morpholinium moieties were investigated to understand their effect on the structure, stability, and activity of two proteins, bovine serum albumin (BSA) and lysozyme (Lyz). In the first objective, we investigated the impact of imidazolium based ILs on the structure and stability of two proteins using different spectroscopic techniques. Our results revealed that hydrophobicity of cationic part of ILs has a significant influence on the destabilization of proteins. Also, the concentration of ILs placed an important role in their stability. In the second objective, few mostly used conventional buffers were investigated on the thermal aggregation behavior of BSA. Aggregation studies revealed that out of three buffers (Phosphate, TRIS, and Imidazole), phosphate buffer ceases the heat induced aggregation. Based on the results, the third objective of this thesis proceeded with different ammonium based ILs. The main outcome was observed to be contradictory to the results obtained in first objective. Here the most hydrophobic IL has a stabilizing effect on the thermally unfolded BSA and also higher concentration has shown a positive effect. However, increasing the hydrophobic nature of the IL, as expected results noticed with the loss of tertiary structure and aggregation at elevated temperature. The last part of this thesis explores the synergistic effect of ILs and polymers (carboxymethylcellulose and polyethyleneglycol) on the activity and structure of Lyz. The results show that all the IL formulations have a protecting effect compared to buffer medium and in some formulations, the activity of Lyz enhanced by ≈ 25%. Overall, this thesis presents the effect of some commonly used ILs as well as some unexplored ILs on the stability and activity of proteins. It also provides an understanding of protein-IL interactions through thermodynamic binding parameters and molecular dynamic simulations. Moreover, the synergistic effect of ILs and polymers recommends further investigation of new formulations based on this in protein studies.