NMR investigations on binding and dynamics of imidazolium-based ionic liquids with HEWL

Molecular level insights on protein-ionic liquid (P-IL) interactions are beneficial for assessing protein stability, binding and dynamics. In the present work, interactions of ILs, namely, 1-butyl 3-methylimidazolium methyl sulfate (IL1), 1-butyl 3-methylimidazolium octyl sulfate (IL2) and 1-butyl 3-methylimidazolium chloride (IL3) with hen egg white lysozyme (HEWL) protein were investigated using solution-state nuclear magnetic resonance (NMR) spectroscopy. To ascertain the binding and dynamics from the perspective of both protein and IL, various ligand based NMR approaches such as selective and non-selective nuclear spin-relaxation (R_1SEL and R_1NS), saturation transfer difference (STD), difference of inversion recovery rate with and without target irradiation (DIRECTION), ³⁵Cl line-shape and spin-relaxation, and protein back bone amide chemical shift perturbations (CSPs) from ¹H-¹⁵N HSQC were utilized. Among the ILs investigated, IL2 experiences significant interaction relative to those of IL1 and IL3, as revealed by the combined R_1SEL and R_1NS analysis, which is further supported by STD NMR. CSP analyses of ¹H-¹⁵N HSQC spectra of aqueous P-IL mixtures enabled to identify the potential binding sites of ILs with HEWL. Whereas, ¹⁵N longitudinal (R₁) and transverse (R₂) spin-relaxation rates and ¹⁵N{¹H} heteronuclear nuclear Overhauser effect (hetNOE) data subjected to the model free analysis for IL2 yielded the rotational correlation times and order parameters of various residues of HEWL. Furthermore, the results could discern the nature of interactions between studied ILs and HEWL in terms of specific and non-specific interactions.