Aromatic vs alicyclic: Hydrophobicity of the ionic liquid on protein stability and fibril formation

Abstract

Ionic liquids (ILs) are environment-friendly solvents with potential applications in biochemical reactions and as pharmaceutical ingredients. They are used to stabilize proteins and act as anti-aggregation agents in which the hydrophobic effect of ILs is suggested to have a primary role. To understand the differences in the effect of aromatic and alicyclic cations of ILs on thermal stability and fibrillation propensity of proteins, the effect of two alicyclic (butyl-1-methylpyrrolidinium and butyl-1-methylpiperidinium) and two aromatic (butyl-4-methylpyridinium and butyl-3-methylimidazolium) ILs on lysozyme was investigated by spectroscopy, densitometry, and molecular dynamics simulation. The results suggest that all the ILs switch the fibrillation pathway from nucleation-independent to nucleation-dependent mechanism at ≥200 mM. The fibrillation is slowed down by 28–35-fold in 500 mM of ILs, exceptionally pyrrolidinium-IL delays the fibrillation by ∼70-fold. All the ILs destabilize the protein during thermal denaturation. Though in all cases the unfolding is enthalpy-driven, pyridinium-IL at higher concentrations exhibits an entropy-driven unfolding effect. The volumetric analysis shows a higher thermal expansion coefficient for Lyz in pyridinium-IL which might be due to the increased internal cavity volume of the protein upon binding of the IL. These results corroborate with the computational studies that the preferential interaction coefficient of aromatic-ILs is more, particularly at a higher concentration (200 mM). Four preferable binding sites for ILs on the protein could be identified that includes the amyloidogenic regions, β-hairpin and the hydrophobic cluster 5. Also, the ILs are found to preferably interact with the protein through their alicyclic/aromatic ring and alkyl sidechain. These results suggest that the binding of ILs to the amyloidogenic region inhibits the fibril formation which has a similar effect for both alicyclic and aromatic ILs whereas aromatic-ILs show a larger destabilization effect than alicyclic-ILs at higher concentrations.