Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein

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 int eraction between MT and NO in live cells, we generated a fusion protein con sisting of MT sandwiched between two mutant green fluorescent proteins (GFP s). In vitro studies with this chimera (FRET-MT) demonstrate that fluoresce nt resonance energy transfer (FRET) can be used to follow conformational ch anges indicative of metal release from MT. Imaging experiments with live en dothelial cells show that agents that increase cytoplasmic Ca2+ act via end ogenously generated NO to rapidly and persistently release metal from MT, A role for this interaction in intact tissue is supported by the finding tha t the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT-/-) unless endogenous NO synthesis is blocked. These results are the fi rst application of intramolecular green fluorescent protein (GFP)-based FRE T in a native protein and demonstrate the utility of FRET-MT as an intracel lular surrogate indicator of NO production, In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is reve aled.