Fluorescence resonance energy transfer-based assays for the real-time detection of nitric oxide signaling

Low-molecular-weight S-nitrosothiols are found in many tissues and affect an array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of the membrane permeant S-nitrosothiol, S-nitrosocysteine ethyl ester (SNCEE), on a fluorescence resonance energy transfer (FRET) reporter based on the cysteine-rich heavy metal binding protein, metallothionein (FRET-MT) flanked by enhanced cyan and yellow fluorescent proteins (ECFP and EYFP, respectively). We previously showed that FRET can be used to follow metal binding and release by this construct. SNCEE (50 microM) induced a decrease in energy transfer, as shown by an increase in the peak emission intensity of the donor fluorophore (ECFP) and a decrease in that of the acceptor (EYFP). These changes in intramolecular FRET were reversed by 50 microM dithiothreitol (DTT), suggesting nitrosothiol-mediated modification of a cysteine residue in MT. Furthermore, the effects of SNCEE on the FRET-MT reporter were not affected by HbO(2), which would be expected to block any process involving (*)NO liberated by decomposition of nitrosothiol but would not necessarily affect transnitrosation. In further support of SNCEE-induced conformational changes in MT, we used live cell imaging of the zinc-sensitive fluorescent indicator FluoZin-3 to show that SNCEE also caused increases in labile Zn(2+).