School of Science, STEM College, RMIT University, Melbourne, VIC 3000, Australia.
La Trobe Institute for Molecular Science, Department of Mathematical and Physical Sciences, School of Computing Engineering and Mathematical Science, La Trobe University, Bundoora, VIC 3086, Australia.
Int J Biol Macromol. 2023 Dec 31;253(Pt 7):127456. doi: 10.1016/j.ijbiomac.2023.127456. Epub 2023 Oct 14.
Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-C-3-methylimidazolium chloride ionic liquid (IL) series ([Cmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore fluorescence, particularly in long alkyl chain ILs ([Cmim]Cl and [Cmim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [Cmim]Cl and [Cmim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [Cmim]Cl and [Cmim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [Cmim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.
绿色荧光蛋白（GFP）及其变体广泛应用于医学和生物学研究，特别是作为蛋白质结构完整性、蛋白质-蛋白质相互作用以及生物传感器的指示剂。本研究采用超折叠 GFP（sfGFP）来研究不同长度的 1- 碳-3-甲基咪唑氯离子液体（IL）系列（[Cmim]Cl，n=2、4、6、8、10、12）对蛋白质荧光、结构、水合、聚集动力学和结晶行为的影响。结果表明，sfGFP 发色团荧光随浓度呈下降趋势，特别是在长烷基链 ILs（[Cmim]Cl 和 [Cmim]Cl）中。色氨酸（Trp）荧光表明，随着烷基链的增长，猝灭速率增加，表明 Trp57 与烷基链之间存在非极性相互作用。在 [Cmim]Cl 和 [Cmim]Cl 的高 IL 浓度 1.5 M 下观察到二级结构变化。小角 X 射线散射（SAXS）表明蛋白质尺寸相对稳定，但在 [Cmim]Cl 和 [Cmim]Cl 溶液中存在 IL 聚集体。动态光散射（DLS）数据显示，随着烷基链 ILs 的增长，蛋白质尺寸和聚集度增加。值得注意的是，除了 [Cmim]Cl 以外的 ILs 和盐促进了 sfGFP 的结晶。本研究强调了阳离子烷基链长度和浓度对蛋白质稳定性和聚集的影响，为利用 IL 溶剂进行蛋白质稳定和结晶目的提供了深入了解。
