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[Display omitted] Lipid-based lyotropic liquid crystalline nanoparticles (LCNPs) face stability challenges in biological fluids during clinical translation. Ionic Liquids (ILs) have emerged as effective solvent additives for tuning the structure of LCNP's and enhancing their stability. We investigated the effect of a library of 21 choline-based biocompatible ILs with 9 amino acid anions as well as 10 other organic/inorganic anions during the preparation of phytantriol (PHY)-based LCNPs, followed by incubation in human serum and serum proteins. Small angle X-ray scattering (SAXS) results show that the phase behaviour of the LCNPs depends on the IL concentration and anion structure. Incubation with human serum led to a phase transition from the inverse bicontinuous cubic (Q 2) to the inverse hexagonal (H 2) mesophase, influenced by the specific IL present. Liquid chromatography-mass spectrometry (LC-MS) and proteomics analysis of selected samples, including PHY control and those with choline glutamate, choline hexanoate, and choline geranate, identified abundant proteins in the protein corona, including albumin, apolipoproteins, and serotransferrin. The composition of the protein corona varied among samples, shedding light on the intricate interplay between ILs, internal structure and surface chemistry of LCNPs, and biological fluids. [ABSTRACT FROM AUTHOR]

[Display omitted] Globular proteins are well-folded model proteins, where ions can substantially influence their structure and aggregation. Ionic liquids (ILs) are salts in the liquid state with versatile ion combinations. Understanding the IL effect on protein behavior remains a major challenge. Here, we employed small angle X-ray scattering to investigate the effect of aqueous ILs on the structure and aggregation of globular proteins, namely, hen egg white lysozyme (Lys), human lysozyme (HLys), myoglobin (Mb), β-lactoglobulin (βLg), trypsin (Tryp) and superfolder green fluorescent protein (sfGFP). The ILs contain ammonium-based cations paired with the mesylate, acetate or nitrate anion. Results showed that only Lys was monomeric, whereas the other proteins formed small or large aggregates in buffer. Solutions with over 17 mol% IL resulted in significant changes in the protein structure and aggregation. The Lys structure was expanded at 1 mol% but compact at 17 mol% with structural changes in loop regions. HLys formed small aggregates, with the IL effect similar to Lys. Mb and βLg mostly had distinct monomer and dimer distributions depending on IL type and IL concentration. Complex aggregation was noted for Tryp and sfGFP. While the anion had the largest ion effect, changing the cation also induced the structural expansion and protein aggregation. [ABSTRACT FROM AUTHOR]

[Display omitted] Proteins generally tend to aggregate with less desirable properties in numerous solvents, which is one of the major challenges in the development of solvents for functional proteins. This work aims to utilize fluorescence spectroscopy and small angle X-ray scattering (SAXS) to understand the effects of ionic liquids (ILs) on the fluorescence and aggregation behavior of superfolder green fluorescent protein (sfGFP). The studied ILs consisted of four different anions coupled with primary, tertiary and quaternary ammonium cations. The results show that the chromophore fluorescence was generally maintained in 1 mol% IL-water mixtures, then decreased with increasing IL concentration. We primarily employed the pseudo-radius of gyration (pseudo-R g) to evaluate sfGFP aggregation. The sfGFP was less aggregated with nitrate-based ILs compared to in buffer, and more aggregated in the mesylate-based ILs. Further, we show that the polyol additives of glycerol and glucose in IL-water mixtures slightly decreased the sfGFP propensity to aggregate. Size-exclusion chromatography (SEC)-SAXS was used to characterize the monomeric sfGFP in ethylammonium nitrate (EAN) and triethylammonium mesylate (TEAMs)-water mixtures. The presence of 1 mol% TEAMs maintained the sfGFP fluorescence, promoted the compact structure, but slightly increased the amount of large aggregates, which contrasted with that of EAN. [ABSTRACT FROM AUTHOR]

Solvents that support protein functionality are important for biochemical applications, and new solvents are required. Here we employ FTIR and fluorescence spectroscopies, small angle X-ray scattering (SAXS) and X-ray crystallography to understand conformational changes of lysozyme with ionic liquids (ILs) added. Spectroscopic techniques identified that the secondary structure of lysozyme was maintained at the lower IL concentrations of 1 and 5 mol%, though the Tryptophan environment was significantly altered with nitrate-based ILs present. SAXS experiments indicated that the radius of gyration of lysozyme increased with 1 mol% IL present, and then decreased with increasing IL concentrations. The tertiary structure, particularly the loop regions, changed as a function of IL concentration, and this depended on the IL type. The crystallographic structure of lysozyme with the IL of ethylammonium nitrate present confirmed the loop region was extended, and identified three specific binding sites with nitrate ions, and that the positively charged areas were IL sensitive regions. This work provides a detailed understanding of lysozyme conformational changes in the presence of ILs. This approach can be extended to other functionally-important proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] Understanding protein behaviour is crucial for developing functional solvent systems. Ionic liquids (ILs) are designer salts with versatile ion combinations, where some suppress unfavourable protein behaviour. This work utilizes small angle X-ray scattering (SAXS) to investigate the size and shape changes of model protein hen egg white lysozyme (HEWL) in 137 IL and salt solutions. Guinier, Kratky, and pair distance distribution analysis were used to evaluate the protein size, shape, and aggregation changes in these solvents. At low IL and salt concentration (1 mol%), HEWL remained monodispersed and globular. Most ILs increased HEWL size compared to buffer, while the nitrate and mesylate anions induced the most significant size increases. IL cation branching, hydroxyl groups, and longer alkyl chains counteracted this size increase. Common salts exhibited specific ion effects, while the IL effect varied with concentration due to complex ion-pairing. Protein aggregation and unfolding occurred at 10 mol% IL, altering the protein shape, especially for ILs with multiple alkyl chains on the cation, or with a mesylate/nitrate anion. This study highlights the usefulness of adopting a high-throughput SAXS strategy for understanding IL effects on protein behaviour and provides insights on controlling protein aggregation and unfolding with ILs. [ABSTRACT FROM AUTHOR]