Recovery of enzyme structure and activity following rehydration from ionic liquid
Recovery of enzyme structure and activity following rehydration from ionic liquid
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从离子液体再水化后酶结构和活性的恢复
DOI：
10.1039/d2cp00608a
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发表时间：
2022
影响因子：
3.3
通讯作者：
Matysiak, Silvina
中科院分区：
文献类型：
--
作者：
Lee, Pei-Yin;Singh, Onkar;Bermudez, Harry;Matysiak, Silvina
在室温下长期保存蛋白质仍然是一个重大挑战。为了使用离子液体（IL）来解决这一挑战，在这里，我们提出了一个实验和模拟的组合，以研究溶菌酶的变化后，从IL混合物使用两个咪唑基IL（1-乙基-3-甲基咪唑乙基硫酸盐，[EMM][EtSO 4]和1-乙基-3-甲基咪唑二乙基磷酸盐，[EMM][Et 2 PO 4]）再水化。通过各种光谱实验和分子动力学模拟来确定溶菌酶的结构和活性。圆二色性光谱证实，即使在295天后，溶菌酶在再水化后仍保持其二级结构。增加IL浓度会降低溶菌酶的活性，并最终在足够高的IL浓度下被淬灭，但来自高IL浓度的溶菌酶的再水化完全恢复其活性。这种再水化发生在最常见的溶菌酶活性测定中，但如果不仔细注意，这种对IL浓度的影响可能会被忽略。从模拟中，我们观察到占领的[EMIM+]离子附近的活性位点和配体-溶菌酶复合物是不太稳定的IL的存在下，这导致在溶菌酶活性的降低。再水化后，观察到[EMIM+]快速离开，活性位点的可用性恢复。此外，在高IL浓度下也观察到结构波动的抑制，这也解释了活性的降低。这种结构抑制在经历再水化后恢复。天然蛋白质结构和活性的恢复表明再水化后溶菌酶恢复到其原始状态。我们的研究结果还提出了一个简单的路线，延长储存后的蛋白质回收。
Long-term preservation of proteins at room temperature continues to be a major challenge. Towards using ionic liquids (ILs) to address this challenge, here we present a combination of experiments and simulations to investigate changes in lysozyme upon rehydration from IL mixtures using two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO4] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et2PO4]). Various spectroscopic experiments and molecular dynamics simulations are performed to ascertain the structure and activity of lysozyme. Circular dichroism spectroscopy confirms that lysozyme maintains its secondary structure upon rehydration, even after 295 days. Increasing the IL concentration decreases the activity of lysozyme and is ultimately quenched at sufficiently high IL concentrations, but the rehydration of lysozyme from high IL concentrations completely restores its activity. Such rehydration occurs in the most common lysozyme activity assay, but without careful attention, this effect on the IL concentration can be overlooked. From simulations we observe occupation of [EMIM+] ions near the vicinity of the active site and the ligand-lysozyme complex is less stable in the presence of ILs, which results in the reduction of lysozyme activity. Upon rehydration, fast leaving of [EMIM+] is observed and the availability of active site is restored. In addition, suppression of structural fluctuations is also observed when in high IL concentrations, which also explains the decrease of activity. This structure suppression is recovered after undergoing rehydration. The return of native protein structure and activity indicates that after rehydration lysozyme returns to its original state. Our results also suggest a simple route to protein recovery following extended storage.