Dissolving Amyloid Fibrils with Natural Deep Eutectic Solvents: Citric Acid−Glycerol Achieves Superior Solubilization and Partial Protein Refolding

Amyloid protein aggregates pose a significant challenge due to their extreme insolubility and association with neurodegenerative diseases. To identify effective solvents for dissolving amyloid-forming proteins, a screening of deep eutectic solvents (DESs), including natural DESs (NADES), and related solvents was conducted. Aggregated lysozyme (LA) was reproducibly prepared under acidic and thermal conditions as a model of poorly soluble protein aggregates. Spectroscopic analyses using Thioflavin T fluorescence and circular dichroism (CD), along with morphological observations under polarized light and SEM, confirmed the formation of β-sheet-rich amyloid-like structures. The citric acid-glycerol DES showed the highest solubility for LA, outperforming other DESs and DMSO. Its performance was further enhanced by the addition of ∼5% water. This result also suggests a strong contribution of multivalent organic acids, such as citric and malic acid, to protein solubilization. CD analysis indicated that lysozyme recovered a native-like secondary structure, suggesting partial structural recovery after dissolution in this DES. These findings highlight the potential of DESs as effective media for dissolving protein aggregates and suggest their possible utility in protein refolding and related biotechnological applications, including potential future biomedical applications.