Conformational Stability of α-Lactalbumin and Lysozyme in Ammonium-Based Ionic Liquids: A Comprehensive Insight into Structure–Activity Relationship

This work investigates the conformational stability of α-lactalbumin (α-LA) and lysozyme (Lys) in the presence of ammonium-based ionic liquids (ILs), such as N-butyl-pyridinium bromide ([BPy][Br]) and 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) with varying aromatic residues. The spectroscopic studies indicated that [BPy][Br] could bind strongly with α-LA and induced conversion of the α-helix to a more β-sheet like structure via H-bonding, charge-pairing, and π-stacking interactions with aromatic amino acid residues (also verified by molecular docking studies). Moreover, the effects of the pH, temperature, concentration, and presence of ionic electrolytes on the binding isotherm were investigated in detail. Unlike α-LA, Lys appeared to be more resistant to IL-induced interactions, maintaining its 3D-structure and optical properties over a wider IL concentration range. These differences stem from alterations in protein surface charge, secondary structure, and IL-protein interactions, particularly the ability of ILs to disrupt hydrogen bonding and hydrophobic interactions. Interestingly, [Bmim][Br] despite its better mobility and ion transport property showed poor interaction with α-LA as compared to [BPy][Br]. This could possibly be due to larger hydrophobicity and superior π-stacking ability of pyridinium rings than that of imidazolium units with amino acid residues. These findings provide insights into protein-IL interactions, with implications for protein engineering, biocatalysis, and biopharmaceutical formulation.