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.

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芳香族与脂环族：离子液体的疏水性对蛋白质稳定性和原纤维形成的影响

离子液体（IL）是环境友好型溶剂，在生化反应和药物成分方面具有潜在的应用前景。它们用于稳定蛋白质并充当抗聚集剂，其中 IL 的疏水作用被认为起主要作用。为了了解离子液体的芳香族和脂环族阳离子对蛋白质热稳定性和原纤维化倾向的影响的差异，两种脂环族（丁基-1-甲基吡咯烷鎓和丁基-1-甲基哌啶鎓）和两种芳香族（丁基-4-甲基吡啶鎓）的影响通过光谱学、光密度测定和分子动力学模拟研究了溶菌酶上的ILs（丁基-3-甲基咪唑鎓和丁基-3-甲基咪唑鎓）。结果表明，所有 IL 在 ≥200 mM 时都会将纤维颤动途径从成核无关机制转变为成核依赖性机制。在 500 mM IL 中，纤维颤动减慢 28-35 倍，特别是吡咯烷鎓-IL 可将纤维颤动延迟约 70 倍。所有 IL 都会在热变性过程中破坏蛋白质的稳定性。尽管在所有情况下，解折叠都是由焓驱动的，但较高浓度的吡啶鎓-IL 表现出熵驱动的解折叠效应。体积分析显示吡啶鎓-IL 中 Lyz 的热膨胀系数较高，这可能是由于与 IL 结合后蛋白质内腔体积增加所致。这些结果证实了芳香族离子液体的优先相互作用系数更大，特别是在较高浓度（200 mM）下的计算研究。可以鉴定蛋白质上 IL 的四个优选结合位点，包括淀粉样蛋白生成区域、β-发夹和疏水簇 5。此外，发现 IL 优先通过其脂环族/芳香环和烷基侧链与蛋白质相互作用。这些结果表明，IL 与淀粉样蛋白生成区域的结合抑制原纤维形成，这对脂环族和芳香族 IL 具有相似的作用，而芳香族 IL 在较高浓度下比脂环族 IL 表现出更大的去稳定作用。