Designer Solvents for Understanding Protein Stability

Proteins have been harnessed for biotechnology applications including but not limited to detergents, pharmaceuticals and biocatalysis. However, many proteins are limited after purification due to their poor long term (shelf life) and thermal stability as well as low solubility in aqueous buffered solutions. This problem can be directly related to the fundamental interactions between proteins and their solvents. Therefore, research into the effect of solvents on protein solubility and stability could shed light on these interactions. A class of non-aqueous solvents showing promise for protein stabilisation is ionic liquids (ILs). ILs are liquids consisting of cations and anions, with tailorable properties due to the structural variation of their ions. Some ILs have been shown to enhance protein solubility, activity and/or stability. However, due to the compounding complexity of IL solvents and protein structure, there is no comprehensive understanding of the influence of ILs on proteins. This necessitates additional, thorough investigation of the many aspects of IL based solvents and protein structure, beginning from a fundamental standpoint of IL structure-property relationships to the specific ion effects of model proteins in IL solvents. This thesis aims to use the characterization of IL physicochemical and solvation properties to better understand their effect on model proteins. Beginning with the synthesis and characterization of novel ILs, followed by the targeted investigation of protic IL (PIL) structural effects on solvation properties. Finally, it aims to correlate the structure and concentration of ILs with their effect on shape, size, and aggregation of the model proteins hen egg white lysozyme (HEWL) and human lysozyme (HL). Initially the experimental aim was to expand the known structure-property relationships of ILs. The use of machine learning (ML) algorithms and the comprehensive experimental characterisation of both novel and reported ILs have been conducted to address this knowledge gap. The work presented herein includes the synthesis and characterization of a library of 50 ILs, systematically chosen for their structural variance. This included structural moieties of hydroxyl groups, primary to quaternary ammonium cations, both inorganic and organic anions as well as varying the alkyl chain length on either the cation or the anion. Each IL was extensively characterized for their physicochemical, thermal, and liquid nanostructure properties. Further analysis using ML algorithms enabled the quantification of the influence of these structural moieties on IL properties, as well as the qualitative prediction of thermal properties. ML showed that density, surface tension and refractive index were almost entirely based on IL structure and the powerful influence of the presence of hydroxyl groups to increase density, surface tension, viscosity, refractive index, glass transition temperature and reduce conductivity. To better understand the interactions between ILs and solutes, the characterisation of Kamlet-Aboud-Taft (KAT) parameters and the ET(30) electronic transition energy scale combined with molecular dynamics (MD) simulations was performed. Within the library of ILs a focus on PILs was made because of their extensive H-bonding capabilities and ease of synthesis. The KAT parameters include π* (dipolarity/polarizability), α (H-bond donating ability) and β (H-bond accepting ability). This revealed that increased alkyl chain length increases β while decreasing π*, α and ET(30). Notably, hydroxyl groups had the opposite effect. The β value of the PIL was shown to be predominantly based on the anion where the nitrate anion was found to be more influential than carboxylate anions for increasing PIL polarizability and reducing β. Radial distribution functions were used to determine atom-atom distances and correlate them with KAT parameters. This showed increased polarizability aligned with greater cation-anion distances and anion-anion distances, and the increase in β correlated to a decrease in cation-cation distances. Next, the previously synthesized library of ILs, as well as common ionic salts, were used to investigate their effect on the model protein HEWL. ILs were used at concentrations of 1, 10, 50 and 100 mol% while the salts were used at 1 mol%. A high energy synchrotron based small angle X-ray scattering (SAXS) method was used for the rapid analysis of samples, which provided structural information on protein size, shape, and aggregation. This enabled a high throughput screening study to correlate IL structural moieties and protein structural stability. The hydroxyl group was highlighted as a potential protein stabilising group at higher IL concentrations, with ethanolammonium acetate, ethanolammonium formate, diethylammonium formate and ethanolammonium propionate supporting non-aggregated HEWL at 50 mol%. Noticeably, an IL concentration of 1 mol% most effectively supported native HEWL structure across almost all ILs while high concentrations of ILs (50 and 100 mol%) often increased HEWL aggregation. Ionic salts at 1 mol% were shown to align with the Hofmeister series while the ILs did not fit within this series due to the complex nature of IL specific ion effects and ion pairing. To further the investigation of specific PILs on protein structures, a study was conducted on the structurally similar proteins HEWL and HL. PILs were used due to their similar H-bonding properties to water, which has caused speculation of their potential protein stabilizing ability. However, the literature involving proteins and ILs has shown ambiguous results when comparing the effect of a PIL across proteins. Therefore, a selection of 5 PILs were chosen from the previous protein study for their varying effects on HEWL stability and used at 1, 10 and 20 mol% in water. Through a combination of size exclusion chromatography (SEC)-SAXS and capillary SAXS, the effects of PIL and protein concentration on protein aggregation were investigated. The effect of PILs was mostly consistent across proteins and despite them increasing protein aggregation as compared to buffer, the 1 mol% PILs were the most effective concentration for supporting monomeric proteins. The chaotropic nature of the nitrate anion was highlighted as both 10 and 20 mol% ethylammonium nitrate had minimal aggregation for either protein. However, SEC analysis highlighted the differences in the aggregation of both proteins in buffer; where HEWL is monomeric, and HL has a small number of aggregates. Overall, this work showed that despite similarities in protein structure, these proteins had significantly different aggregation behaviour.