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

A Spectroscopic and Molecular Simulation Approach toward the Binding Affinity between Lysozyme and Phenazinium Dyes: An Effect on Protein Conformation

A comparative study of binding interaction between Safranin O (SO) and Neutral Red (NR) with lysozyme (Lyz) has been reported using several spectroscopic methods along with computational approaches. Steady-state fluorescence measurements revealed static quenching as the major quenching mechanism in Lyz–SO and Lyz–NR interaction, which is further supported by time-resolved fluorescence and UV–vis measurements. Additionally, binding and thermodynamic parameters of these interactions are calculated from temperature dependent fluorescence data. Moreover, conformational changes of protein upon binding with SO and NR are provided by synchronous and circular dichroism (CD) measurements. Molecular docking study provided the exact binding location of SO and NR in lysozyme. Along with this study, molecular dynamics simulation is carried out to measure the stability of Lyz, Lyz–SO, and Lyz–NR complex. The present study revealed the strong binding affinity of dyes with lysozyme, and this study would be helpful toward medical and environmental science

Impact of imidazolium-based ionic liquids on the structure and stability of lysozyme

<p>Various types of water-miscible aprotic ionic liquids (ILs) with different cations (1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, 1-octyl-3-methylimidazolium) and anions (ethylsulfate and chloride) were used as co-solvents to investigate the stability of lysozyme. Different techniques such as fluorescence, thermal absorption, and circular dichroism (CD) spectroscopy have been used for the study. Fluorescence results reveal that the addition of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium) increases the hydrophobicity around the tryptophan environment in lysozyme. CD analysis and temperature-dependent studies were done to investigate the stability of the protein. From the CD analysis, it was observed that the ILs keep the native structure of protein intact. Thermal denaturation studies depicted that the melting temperature of the protein increased in the presence of ILs (1-ethyl-3-methylimidazolium ethyl sulfate and 1-ethyl-3-methylimidazolium), which indicates the stabilization of the protein.</p