Regulation of protein disaggregation by the hydrophobic chain length of ammonium-based ionic liquids

Protein aggregation and disaggregation are critical in determining biomolecular stability and function. This study explores the influence of ammonium-based ionic liquids with varying hydrophobic chain lengths on the aggregation and disaggregation of bone morphogenetic protein-2 (BMP-2). Thioflavin T (ThT) fluorescence spectroscopy showed a fivefold increase in fluorescence upon aggregation, which further intensified in the presence of N8, indicating enhanced fibrillation. Fourier transform infrared (FTIR) spectroscopy analysis revealed that heat-aggregated BMP-2 exhibited a β-sheet content of 63% ± 9%, which increased to 68% ± 9% upon treatment with N8, whereas shorter-chain ionic liquids (N2 and N4) reduced the β-sheet content to 37% ± 4% and 32% ± 5%, respectively. Dynamic light scattering (DLS) measurements confirmed that the native protein had a hydrodynamic radius of 12 ± 5 nm, which increased to 390 ± 60 nm upon aggregation. Treatment with N8 and N6 further increased the radius to 680 ± 150 nm and 530 ± 100 nm, respectively, whereas N2 and N4 reduced it to 37 ± 5 nm and 60 ± 11 nm, respectively. Cytotoxicity assays indicated increased nuclear fragmentation and reactive oxygen species (ROS) generation with longer-chain ionic liquids. These findings suggest that shorter-chain ammonium-based ionic liquids exhibit greater disaggregating potential owing to the interruption of hydrogen bonds and π-π stacking interactions, which stabilize the fibril structure. Longer chains stabilize fibrils and promote fibrillation owing to the hydrophobic interaction between the alkyl chain of the cation and the hydrophobic surface of aggregates, providing insights into protein stability modulation.