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

The complexation of glutathione (GSH) with divalent cadmium ion has been used as a typical model for investigating the coordination chemistry of sulfydryl-containing peptides and heavy metal ions, which is essential to understand the mechanism of intracellular cadmium detoxification. In this study, the single-molecule reaction between GSH molecule and Cd2+ ion was monitored in real time by a nanoreactor that formed by a mutant (M113R)(7) alpha-hemolysin (alpha HL) protein nanopore equipped with a novel per-6-quaternary ammonium-beta-cyclodextrin (p-QABCD). The reaction pathways, intermediates, and products could be recognized by analyzing the current fluctuations. The reaction between Cd2+ and GSH was highly dependent on solution pH value. Cd(GSH)(2) was the only final product at pH 7.4, while both Cd(GSH)(2) and Cd-2(GSH)(2) were present at pH 9.0. The formation of Cd-2(GSH)(2) follows two possible pathways: (1) one Cd2+ ion first coordinates with the thiol group of two GSH molecules to form Cd(GSH)(2), and then the second Cd2+ ion quickly incorporates with the deprotonated amino group of Cd(GSH)(2) to produce Cd-2(GSH)(2); (2) two Cd2+ ions separately coordinate with the thiol and deprotonated amino group of one GSH molecule to yield Cd-2(GSH)(1), and the second GSH molecule binds Cd2+ ions quickly to form Cd-2(GSH)(2). The free-labeling and free-modifying method for monitoring single-molecule chemical reaction was simple and sensitive, which would be important to further understand intracellular mechanisms of detoxification of heavy metals. This work greatly expands the research field of single-molecule nanopore technique.