A biophysical and computational investigation of aggregation suppression effect of choline dodecyl prolate ionic...

The inherent susceptibility of proteins to aggregation under stress conditions plays a pivotal role in the molecular pathology of neurodegenerative disorders and poses significant challenges in biopharmaceutical stability. Several studies have clearly shown that ionic liquids (IL) have contributed significantly in overcoming these challenges by providing structural stabilization and suppression of aggregate prone conformations in proteins. We have exploited robust biophysical and in silico profiling of biocompatible choline-based IL imparting stability to α-lactalbumin (α-LA) from heat-induced aggregation. Fluorescence-based assays including Th-T, ANS, rayleigh scattering, and intrinsic fluorescence were employed to comprehensively assess conformational changes. Along with these measurements, time resolved fluorescence spectroscopy as well as anisotropy results depicted that IL effectively protected α-LA from aggregation in a concentration-dependent manner. Thermal and secondary structure profiling through DSC and far-UV CD, respectively, revealed increment in thermostability and α-helical content as well as significant suppression of β-sheet enrichment which occurs in aggregated condition. Similarly, DLS and confocal microscopy revealed reduced hydrodynamic diameter and negligible aggregation in the presence of IL, contrasting extensive aggregates in the heated protein. Computational analysis through molecular docking and molecular dynamic simulations further supported strong binding and stabilization of protein-IL complex where integrity of α-LA was maintained via intermolecular forces. Overall, these findings highlight potential of choline-based ILs in preventing protein aggregation and maintaining its structural integrity under stress and extend its applicability in related pathologies, including neurodegenerative disorders.