Fluorescent protein reporters for understanding how glutathione modulates metal interactions

Abstract

Metals are essential cofactors involved in nearly every known biological process. At the same time, transition and heavy metals are widespread environmental toxins that pose a persistent hazard to human health. To understand how metals interact with proteins, and the effect that low molecular weight ligands have on these interactions, we developed the metal-sensitive fluorescent protein msGFP and adapted the redox reporter roGFP2 as sensors for metal interactions with solvent-accessible cysteines. We found that these reporters bound copper, mercury, and lead tightly but had lower affinity for other metals. The inclusion of physiological concentrations of glutathione inhibited these and most other tested metal interactions, in some cases reducing interaction strength by several orders of magnitude. Conversely, we found that glutathione strongly increased the affinity of arsenic and cadmium for the same site. We showed that these interactions are responsive to glutathione in a dose-dependent fashion and used glutathione depletion to replicate this effect inside cells.