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

Abstract

Abstract 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 α-hemolysin (αHL) protein nanopore equipped with a novel per-6-quaternary ammonium-β-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 Cd2(GSH)2 were present at pH 9.0. The formation of Cd2(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 Cd2(GSH)2; (2) two Cd2+ ions separately coordinate with the thiol and deprotonated amino group of one GSH molecule to yield Cd2(GSH)1, and the second GSH molecule binds Cd2+ ions quickly to form Cd2(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.