Insights on lysozyme aggregation in protic ionic liquid solvents by using small angle X-ray scattering and high throughput screening

Abstract

Solvents play a crucial role in protein instability and aggregation, which are undesirable in biomedical applications. The development of quantitative, high throughput methods of evaluating protein aggregation is important in the search for novel solvent mixtures/systems, such as ionic liquids (ILs). In this study, three different small-angle X-ray scattering setups were employed to determine lysozyme size, shape, and aggregation in IL-water mixtures, including a high throughput plate approach, capillary autoloader, and size-exclusion setup, in order of increasing measurement time and scattering data quality. Protein size and shape were determined in low concentrations of ethylammonium nitrate, and the degree of protein aggregation was evaluated by a pseudo-radius of gyration (pseudo-Rg). The short collection time, and wide viscosity tolerance render the plate setup ideal for high throughput screening of protein aggregation in complex IL systems. We then investigated the effect of five additives, known to stabilize proteins, on lysozyme in 144 different IL-water mixtures. We showed that lysozyme lost its shape and had increased aggregation with over 17 mol% IL present, though lysozyme was less aggregated in ethylammonium nitrate (EAN) and ethanolammonium formate (EtAF) solutions. Further, lysozyme aggregation was slightly suppressed by glycerol in EAN-water mixtures, and by trimethylamine N-oxide in EtAF and ethylammonium formate aqueous solutions. This work develops a new quantitative and high throughput approach for evaluating protein aggregation in solvent systems, including viscous solvents.