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Although the function of metallothionein (MT), a 6-to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca 2؉ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT ؊͞؊ ) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed. Metallothioneins (MT) are 6-to 7-kDa intracellular cysteine-rich (30 mol%) metal binding proteins whose function remains elusive (1). A critical role for MT in protection against toxic non-essential metals such as cadmium is apparent (2), and MT appears to act as an antioxidant under a variety of conditions (3). More recently, in vitro data support the hypothesis that MT is a critical link between cellular redox state and metal ion homeostasis (4-6). In this regard, cysteines of metal thiolate clusters confer unique redox sensitivity to an otherwise redox inert metal ligand (e.g., zinc) and facilitate the potential for MT to participate in intracellular signal transduction pathways (7). In the current study, we examine this latter novel hypothesis in intact cells and tissue.We chose to study the interaction of MT and the free radical, nitric oxide (NO), because (i) the bioregulatory targets of NO usually contain cysteines and͞or metals at their active or allosteric site (8); (ii) NO (or a secondary product) reacts with MT in vitro, leading to the release of zinc (9) or cadmium (10); (iii) NO can form stable EPR-detectable complexes with MT in vitro (11); and (iv) MT can reduce the sensitivity of cells to potential toxic levels of NO (12). Application of a chimeric construct (called FRET-MT) based on a recently described cameleon for calmodulin (13) revealed an NO-induced conformational change in MT, indicative of metal release, thereby providing the first demonstrations of (i) changes in intramolecular FRET (fluorescence resonance energy transfer) of a native protein; and (ii) metal release from MT in response to physiologic stimuli in intact cells. Furthermore, the lack of myogen...

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