工程化蛋白质纳米孔实时监测镉离子与谷胱甘肽的单分子反应

Engineered Protein Nanopore for Real-time Monitoring Single-molecule Reaction between Cadmium Ion and Glutathione

本研究利用合成的全-6-季铵基-β-环糊精(Per-6-quaternary ammonium-β-cyclodextrin,p-QABCD)装备基因工程化的α-溶血素(α-Hemolysin,αHL)蛋白质纳米孔(M113R)_7,构建全新的单分子纳米孔反应器,在单分子水平实现对谷胱甘肽(GSH)和镉离子(Cd^(2+))的络合反应的实时原位监测,并辨认络合反应的不同路径、反应中间产物和最终产物。结果表明,溶液的pH值显著影响GSH与Cd^(2+)络合产物的络合比例。pH=7.4时,GSH与Cd^(2+)络合反应的最终产物为Cd(GSH)_2;pH=9.0时,最终产物为Cd(GSH)_2和Cd_2(GSH)_2。其中,Cd_2(GSH)_2的形成遵循两种路径:(1)一个Cd^(2+)首先结合两个GSH分子的巯基形成Cd(GSH)_2,然后,第二个Cd^(2+)结合去质子化的氨基形成Cd_2(GSH)_2;(2)两个Cd^(2+)分别结合同一个GSH分子的巯基和去质子化的氨基形成Cd_2(GSH)_1,然后,第二个GSH分子的巯基和去质子化的氨基结合Cd_2(GSH)_1的Cd^(2+)形成Cd_2(GSH)_2。本研究实现了在单分子水平无标记和无化学修饰研究金属离子和生物小分子的反应,对理解细胞内重金属的解毒机理和拓展纳米孔单分子技术的研究领域具有重要意义。

The chelating reaction between glutathione peptides and divalent cadmium ion was used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions, which was essential to understand the mechanism of intracellular cadmium detoxification. Here, the mutant （CM113R ）7 β HL protein nanopore equipped with a first synthesized per-6 -quaternary ammonium-茁-cyclodextrin （p-QABCD） was used as nanoreactor and detector to investigate the single-molecule reaction of GSH molecules and Cd^2＋ ions. Different reaction pathways, intermediates, and products could be recognized. Cd^2＋- GSH reaction was highly dependent on the solution pH. The Cd（ GSH） 2 was formed at pH 7.4, while the Cd （GSH）2 and Cd2（GSH）2 were formed at pH 9.0. Cd2（GSH）2 was formed by two possible pathways： （1） A Cd^2＋ ion primarily coordinated with the thiol group of two GSH molecules to form Cd（ GSH）2,and then the second Cd^2＋ ion quickly incorporated with the deprotonated amino group of Cd（GSH）2 to form Cd2（GSH）2; （2） Two Cd^2＋ ions separately coordinated with the thiol and deprotonated amino group of one GSH molecule to form Cd2（GSH）lT and the second GSH molecule quickly bounded to Cd^2＋ ions to form Cd2（ GSH）2. This work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification, and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.