Scattering approaches to unravel protein solution behaviors in ionic liquids and deep eutectic solvents: From basic principles to recent developments.

Proteins in ionic liquids (ILs) and deep eutectic solvents (DESs) have gained significant attention due to their potential applications in various fields, including biocatalysis, bioseparation, biomolecular delivery, and structural biology. Scattering approaches including dynamic light scattering (DLS) and small-angle X-ray and neutron scattering (SAXS and SANS) have been used to understand the solution behavior of proteins at the nanoscale and microscale. This review provides a thorough exploration of the application of these scattering techniques to elucidate protein properties in ILs and DESs. Specifically, the review begins with the theoretical foundations of the relevant scattering approaches and describes the essential solvent properties of ILs and DESs linked to scattering such as refractive index, scattering length density, ion-pairs, liquid nanostructure, solvent aggregation, and specific ion effects. Next, a detailed introduction is provided on protein properties such as type, concentration, size, flexibility and structure as observed through scattering methodologies. This is followed by a review of the literature on the use of scattering for proteins in ILs and DESs. It is highlighted that enhanced data analysis and modeling tools are necessary for assessing protein flexibility and structure, and for understanding protein hydration, aggregation and specific ion effects. It is also noted that complementary approaches are recommended for comprehensively understanding the behavior of proteins in solution due to the complex interplay of factors, including ion-binding, dynamic hydration, intermolecular interactions, and specific ion effects. Finally, the challenges and potential research directions for this field are proposed, including experimental design, data analysis approaches, and supporting methods to obtain fundamental understandings of complex protein behavior and protein systems in solution. We envisage that this review will support further studies of protein interface science, and in particular studies on solvent and ion effects on proteins.

Crystal structure via fluctuation scattering.

Crystallography is a quintessential method for determining the atomic structure of crystals. The most common implementation of crystallography uses single crystals that must be of sufficient size, typically tens of micrometres or larger, depending on the complexity of the crystal structure. The emergence of serial data-collection methods in crystallography, particularly for time-resolved experiments, opens up opportunities to develop new routes to structure determination for nanocrystals and ensembles of crystals. Fluctuation X-ray scattering is a correlation-based approach for single-particle imaging from ensembles of identical particles, but has yet to be applied to crystal structure determination. Here, an iterative algorithm is presented that recovers crystal structure-factor intensities from fluctuation X-ray scattering correlations. The capabilities of this algorithm are demonstrated by recovering the structure of three small-molecule crystals and a protein crystal from simulated fluctuation X-ray scattering correlations. This method could facilitate the recovery of structure-factor intensities from crystals in serial crystallography experiments and relax sample requirements for crystallography experiments.

Small-Angle X-ray Scattering Study of the Amphiphilic Bulk Nanostructure of Tetraalkylammonium Deep Eutectic Solvents.

Deep eutectic solvents (DESs) are low-melting mixtures, often prepared from a salt and a molecular hydrogen bond donor. Like ionic liquids, DESs that contain at least one sufficiently amphiphilic component can form bicontinuous nanostructures consisting of polar and nonpolar domains, although this has not been widely explored for many DES combinations. Here, the bulk nanostructures of DESs comprising tetraalkylammonium bromide salts (tetrabutylammonium bromide, tetraoctylammonium bromide, and methyltrioctylammonium bromide) with alkanols and alkanoic acids of systematically varied chain lengths (C2, C6, C8, and C10) as hydrogen bond donors have been studied. Small-angle X-ray scattering techniques were used to identify the relationship between the alkyl chain length and functionality of the hydrogen bond donor on the nature of the amphiphilic nanostructures formed. These findings demonstrated that the amphiphilic nanostructures of the DESs were not affected by the functional group on the hydrogen bond donor, with these nanostructures influenced primarily by both the absolute and relative alkyl chain lengths of the salt and hydrogen bond donor.

Thermal Stability of Protic Ionic Liquids.

While protic ionic liquids (ILs) have found great success as solvents for a broad range of applications, little is known about their degradation when exposed to temperatures above ambient for extended periods of time. Here, we report the thermal stability of six protic ILs, namely, ethylammonium nitrate, ethylammonium formate, ethylammonium acetate, ethanolammonium nitrate, ethanolammonium formate, and ethanolammonium acetate. The effect of heating each ionic liquid to 60 °C for 1 h or 1 week (sealed or open to the atmosphere) was evaluated by considering the changes to water content, pH, mass, thermal phase transitions, and molecular structure after each treatment. Heating each of the six ILs when sealed led to measurable shifts in their water content and 10 wt % pH, but there was no significant change in their mass, thermal phase transitions according to differential scanning calorimetry (DSC), or molecular structure using proton nuclear magnetic resonance (1H NMR) spectra, indicating that the samples were largely unchanged. The samples that were heated open to the atmosphere also displayed no significant changes after 1 h but displayed significant changes after 1 week.

Deep Eutectic Solvent Eutectogels for Delivery of Broad-Spectrum Antimicrobials.

Gel-based wound dressings have gained popularity within the healthcare industry for the prevention and treatment of bacterial and fungal infections. Gels based on deep eutectic solvents (DESs), known as eutectogels, provide a promising alternative to hydrogels as they are non-volatile and highly tunable and can solubilize therapeutic agents, including those insoluble in hydrogels. A choline chloride:glycerol-cellulose eutectogel was loaded with numerous antimicrobial agents including silver nanoparticles, black phosphorus nanoflakes, and commercially available pharmaceuticals (octenidine dihydrochloride, tetracycline hydrochloride, and fluconazole). The eutectogels caused >97% growth reduction in Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria and the fungal species Candida albicans.

Biophysical Characterization and Cryopreservation of Mammalian Cells Using Ionic Liquids.

Ionic liquids (ILs) are a diverse class of solvents which can be selected for task-specific properties, making them attractive alternatives to traditional solvents. To tailor ILs for specific biological applications, it is necessary to understand the structure-property relationships of ILs and their interactions with cells. Here, a selection of carboxylate anion-based ILs were investigated as cryoprotectants, which are compounds added to cells before freezing to mitigate lethal freezing damage. The cytotoxicity, cell permeability, thermal behavior, and cryoprotective efficacy of the ILs were assessed with two model mammalian cell lines. We found that the biophysical interactions, including permeability of the ILs, were influenced by considering the IL pair together, rather than as single species acting independently. All of the ILs tested had high cytotoxicity, but ethylammonium acetate demonstrated good cryoprotective efficacy for both cell types tested. These results demonstrate that despite toxicity, ILs may be suitable for certain biological applications. It also demonstrates that more research is required to understand the contribution of ion pairs to structure-property relationships and that knowing the behavior of a single ionic species will not necessarily predict its behavior as part of an IL.

Lyotropic liquid crystal phases of monoolein in protic ionic liquids.

Monoolein-based liquid crystal phases are established media that are researched for various biological applications, including drug delivery. While water is the most common solvent for self-assembly, some ionic liquids (ILs) can support lipidic self-assembly. However, currently, there is limited knowledge of IL-lipid phase behavior in ILs. In this study, the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly, namely ethylammonium nitrate, ethanolammonium nitrate, ethylammonium formate, ethanolammonium formate, ethylammonium acetate, and ethanolammonium acetate. These ILs were selected to identify specific ion effects on monoolein self-assembly, specifically increasing the alkyl chain length of the cation or anion, the presence of a hydroxyl group in the cation, and varying the anion. The lyotropic liquid crystal phases with 20-80 wt. % of monoolein were characterized over a temperature range from 25 to 65 °C using synchrotron small angle x-ray scattering and cross-polarized optical microscopy. These results were used to construct partial phase diagrams of monoolein in each of the six protic ILs, with inverse hexagonal, bicontinuous cubic, and lamellar phases observed. Protic ILs containing the ethylammonium cation led to monoolein forming lamellar and bicontinuous cubic phases, while those containing the ethanolammonium cation formed inverse hexagonal and bicontinuous cubic phases. Protic ILs containing formate and acetate anions favored bicontinuous cubic phases across a broader range of protic IL concentrations than those containing the nitrate anion.

Regulating the structural polymorphism and protein corona composition of phytantriol-based lipid nanoparticles using choline ionic liquids.

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 (Q2) to the inverse hexagonal (H2) 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.

Structure, aggregation dynamics and crystallization of superfolder green fluorescent protein: Effect of long alkyl chain imidazolium ionic liquids.

Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-Cn-3-methylimidazolium chloride ionic liquid (IL) series ([Cnmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore fluorescence, particularly in long alkyl chain ILs ([C10mim]Cl and [C12mim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [C10mim]Cl and [C12mim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [C10mim]Cl and [C12mim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [C2mim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.

Probing ion-binding at a protein interface: Modulation of protein properties by ionic liquids.

Ions are important to modulate protein properties, including solubility and stability, through specific ion effects. Ionic liquids (ILs) are designer salts with versatile ion combinations with great potential to control protein properties. Although protein-ion binding of common metals is well-known, the IL effect on proteins is not well understood. Here, we employ the model protein lysozyme in dilute and concentrated IL solutions to determine the specific ion binding effect on protein phase behaviour, activity, size and conformational change, aggregation and intermolecular interactions. A combination of spectroscopic techniques, activity assays, small-angle X-ray scattering, and crystallography highlights that ILs, particularly their anions, bind to specific sites in the protein hydration layer via polar contacts on charged, polar and aromatic residues. The specific ion binding can induce more flexible loop regions in lysozyme, while the ion binding in the bulk phase can be more dynamic in solution. Overall, the protein behaviour in ILs depends on the net effect of nonspecific interactions and specific ion binding. Compared to formate, the nitrate anion induced high protein solubility, low activity, elongated shape and aggregation, which is largely owing to its higher propensity for ion binding. These findings provide new insights into protein-IL binding interactions and using ILs to modulate protein properties.

Phytantriol phase behaviour in choline chloride urea and water mixtures.

Deep eutectic solvents (DES) are tailorable non-aqueous solvents with promising properties for a range of applications, from industrial dissolution of plant products to biomedicine. They are mixtures of hydrogen bond donors and acceptors with low melting points that can be tailored to specific applications, and many support the self-assembly of amphiphilic molecules into lyotropic liquid crystal phases. Self-assembled lipid structures have potential for numerous applications, including drug delivery. These ordered structures can act as carriers, slow-release vehicles, or microreactors. Lipid self-assembly in non-aqueous solvents, such as deep eutectic solvents, is important for applications at extreme temperatures, or involving water-insoluble or water sensitive components. However, lipid self-assembly in these solvents remains largely unexplored. In this paper, we have examined the self-assembly of phytantriol, a non-ionic lipid, at 10 and 30 wt% in the deep eutectic solvent choline chloride:urea, with and without water. Self-assembly was assessed using small angle X-ray scattering and cross polarised optical microscopy at temperatures from 25-66 °C. We found that pure choline chloride:urea supports a Pn3m cubic phase similar to that formed in water. However, mixtures of the DES with water resulted in phytantriol forming an inverse hexagonal phase and influenced the phase transition temperatures. These results demonstrate that choline chloride:urea can support diverse phase behaviour, and also provides a mechanism for tailoring the phase for particular applications simply by controlling the amount of water in the solvent. In the future this could lead to methods of triggered release of drugs and biomolecules by the simple addition of water which could be critical for drug delivery applications.

Small angle X-ray scattering investigation of ionic liquid effect on the aggregation behavior of globular proteins.

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), ß-lactoglobulin (ßLg), 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 ßLg 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.

Automation of liquid crystal phase analysis for SAXS, including the rapid production of novel phase diagrams for SDS-water-PIL systems.

Lyotropic liquid crystal phases (LCPs) are widely studied for diverse applications, including protein crystallization and drug delivery. The structure and properties of LCPs vary widely depending on the composition, concentration, temperature, pH, and pressure. High-throughput structural characterization approaches, such as small-angle x-ray scattering (SAXS), are important to cover meaningfully large compositional spaces. However, high-throughput LCP phase analysis for SAXS data is currently lacking, particularly for patterns of multiphase mixtures. In this paper, we develop semi-automated software for high throughput LCP phase identification from SAXS data. We validate the accuracy and time-savings of this software on a total of 668 SAXS patterns for the LCPs of the amphiphile hexadecyltrimethylammonium bromide (CTAB) in 53 acidic or basic ionic liquid derived solvents, within a temperature range of 25-75 °C. The solvents were derived from stoichiometric ethylammonium nitrate (EAN) or ethanolammonium nitrate (EtAN) by adding water to vary the ionicity, and adding precursor ions of ethylamine, ethanolamine, and nitric acid to vary the pH. The thermal stability ranges and lattice parameters for CTAB-based LCPs obtained from the semi-automated analysis showed equivalent accuracy to manual analysis, the results of which were previously published. A time comparison of 40 CTAB systems demonstrated that the automated phase identification procedure was more than 20 times faster than manual analysis. Moreover, the high throughput identification procedure was also applied to 300 unpublished scattering patterns of sodium dodecyl-sulfate in the same EAN and EtAN based solvents in this study, to construct phase diagrams that exhibit phase transitions from micellar, to hexagonal, cubic, and lamellar LCPs. The accuracy and significantly low analysis time of the high throughput identification procedure validates a new, rapid, unrestricted analytical method for the determination of LCPs.

Deep eutectic solvents as cryoprotective agents for mammalian cells.

Cryopreservation has facilitated numerous breakthroughs including assisted reproductive technology, stem cell therapies, and species preservation. Successful cryopreservation requires the addition of cryoprotective agents to protect against freezing damage and dehydration. For decades, cryopreservation has largely relied on the same two primary agents: dimethylsulfoxide and glycerol. However, both of these are toxic which limits their use for cells destined for clinical applications. Furthermore, these two agents are ineffective for hundreds of cell types, and organ and tissue preservation has not been achieved. The research presented here shows that deep eutectic solvents can be used as cryoprotectants. Six deep eutectic solvents were explored for their cryoprotective capacity towards mammalian cells. The solvents were tested for their thermal properties, including glass transitions, toxicity, and permeability into mammalian cells. A deep eutectic solvent made from proline and glycerol was an effective cryoprotective agent for all four cell types tested, even with extended incubation prior to freezing. This deep eutectic solvent was more effective and less toxic than its individual components, highlighting the importance of multi-component systems. Cells were characterised post-thawing using atomic force microscopy and confocal microscopy. Molecular dynamics simulations support the biophysical parameters obtained by experimentation. This is one of the first times that this class of solvents has been systematically tested for cryopreservation of mammalian cells and as such this research opens the way for the development of potentially thousands of new cryoprotective agents that can be tailored to specific cell types. The demonstrated capacity of cells to be incubated with the deep eutectic solvent at 37 °C for hours prior to freezing without significant loss of viability is a major step toward the storage of organs and tissues.

Observations of phase changes in monoolein during high viscous injection.

Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.

Machine learning investigation of viscosity and ionic conductivity of protic ionic liquids in water mixtures.

Ionic liquids (ILs) are well classified as designer solvents based on the ease of tailoring their properties through modifying the chemical structure of the cation and anion. However, while many structure-property relationships have been developed, these generally only identify the most dominant trends. Here, we have used machine learning on existing experimental data to construct robust models to produce meaningful predictions across a broad range of cation and anion chemical structures. Specifically, we used previously collated experimental data for the viscosity and conductivity of protic ILs [T. L. Greaves and C. J. Drummond, Chem. Rev. 115, 11379-11448 (2015)] as the inputs for multiple linear regression and neural network models. These were then used to predict the properties of all 1827 possible cation-anion combinations (excluding the input combinations). These models included the effect of water content of up to 5 wt. %. A selection of ten new protic ILs was then prepared, which validated the usefulness of the models. Overall, this work shows that relatively sparse data can be used productively to predict physicochemical properties of vast arrays of ILs.

Preferred orientation and its effects on intensity-correlation measurements.

Intensity-correlation measurements allow access to nanostructural information on a range of ordered and disordered materials beyond traditional pair-correlation methods. In real space, this information can be expressed in terms of a pair-angle distribution function (PADF) which encodes three- and four-body distances and angles. To date, correlation-based techniques have not been applied to the analysis of microstructural effects, such as preferred orientation, which are typically investigated by texture analysis. Preferred orientation is regarded as a potential source of error in intensity-correlation experiments and complicates interpretation of the results. Here, the theory of preferred orientation in intensity-correlation techniques is developed, connecting it to the established theory of texture analysis. The preferred-orientation effect is found to scale with the number of crystalline domains in the beam, surpassing the nanostructural signal when the number of domains becomes large. Experimental demonstrations are presented of the orientation-dominant and nanostructure-dominant cases using PADF analysis. The results show that even minor deviations from uniform orientation produce the strongest angular correlation signals when the number of crystalline domains in the beam is large.

Electrochemical Stability of Zinc and Copper Surfaces in Protic Ionic Liquids.

Ionic liquids are versatile solvents that can be tailored through modification of the cation and anion species. Relatively little is known about the corrosive properties of protic ionic liquids. In this study, we have explored the corrosion of both zinc and copper within a series of protic ionic liquids consisting of alkylammonium or alkanolammonium cations paired with nitrate or carboxylate anions along with three aprotic imidazolium ionic liquids for comparison. Electrochemical studies revealed that the presence of either carboxylate anions or alkanolammonium cations tend to induce a cathodic shift in the corrosion potential. The effect in copper was similar in magnitude for both cations and anions, while the anion effect was slightly more pronounced than that of the cation in the case of zinc. For copper, the presence of carboxylate anions or alkanolammonium cations led to a notable decrease in corrosion current, whereas an increase was typically observed for zinc. The ionic liquid-metal surface interactions were further explored for select protic ionic liquids on copper using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) to characterize the interface. From these studies, the oxide species formed on the surface were identified, and copper speciation at the surface linked to ionic liquid and potential dependent surface passivation. Density functional theory and ab initio molecular dynamics simulations revealed that the ethanolammonium cation was more strongly bound to the copper surface than the ethylammonium counterpart. In addition, the nitrate anion was more tightly bound than the formate anion. These likely lead to competing effects on the process of corrosion: the tightly bound cations act as a source of passivation, whereas the tightly bound anions facilitate the electrodissolution of the copper.

Protic Ionic Liquid Cation Alkyl Chain Length Effect on Lysozyme Structure.

Solvents that stabilize protein structures can improve and expand their biochemical applications, particularly with the growing interest in biocatalytic-based processes. Aiming to select novel solvents for protein stabilization, we explored the effect of alkylammonium nitrate protic ionic liquids (PILs)-water mixtures with increasing cation alkyl chain length on lysozyme conformational stability. Four PILs were studied, that is, ethylammonium nitrate (EAN), butylammonium nitrate (BAN), hexylammonium nitrate (HAN), and octylammonium nitrate (OAN). The surface tension, viscosity, and density of PIL-water mixtures at low to high concentrations were firstly determined, which showed that an increasing cation alkyl chain length caused a decrease in the surface tension and density as well as an increase in viscosity for all PIL solutions. Small-angle X-ray scattering (SAXS) was used to investigate the liquid nanostructure of the PIL solutions, as well as the overall size, conformational flexibility and changes to lysozyme structure. The concentrated PILs with longer alkyl chain lengths, i.e., over 10 mol% butyl-, 5 mol% hexyl- and 1 mol% octylammonium cations, possessed liquid nanostructures. This detrimentally interfered with solvent subtraction, and the more structured PIL solutions prevented quantitative SAXS analysis of lysozyme structure. The radius of gyration (Rg) of lysozyme in the less structured aqueous PIL solutions showed little change with up to 10 mol% of PIL. Kratky plots, SREFLEX models, and FTIR data showed that the protein conformation was maintained at a low PIL concentration of 1 mol% and lower when compared with the buffer solution. However, 50 mol% EAN and 5 mol% HAN significantly increased the Rg of lysozyme, indicating unfolding and aggregation of lysozyme. The hydrophobic interaction and liquid nanostructure resulting from the increased cation alkyl chain length in HAN likely becomes critical. The impact of HAN and OAN, particularly at high concentrations, on lysozyme structure was further revealed by FTIR. This work highlights the negative effect of a long alkyl chain length and high concentration of PILs on lysozyme structural stability.

Protein aggregation and crystallization with ionic liquids: Insights into the influence of solvent properties.

Ionic liquids (ILs) have been used in solvents for proteins in many applications, including biotechnology, pharmaceutics, and medicine due to their tunable physicochemical and biological properties. Protein aggregation is often undesirable, and predominantly occurs during bioprocesses, while the aggregation process can be reversible or irreversible and the aggregates formed can be native/non-native and soluble/insoluble. Recent studies have clearly identified key properties of ILs and IL-water mixtures related to protein performance, suggesting the use of the tailorable properties of ILs to inhibit protein aggregation, to promote protein crystallization, and to control protein aggregation pathways. This review discusses the critical properties of IL and IL-water mixtures and presents the latest understanding of the protein aggregation pathways and the development of IL systems that affect or control the protein aggregation process. Through this feature article, we hope to inspire further advances in understanding and new approaches to controlling protein behavior to optimize bioprocesses.