ESTABLISHMENT OF 2 DISTINCT PROTEIN DOMAINS IN BLUE-CRAB CALLINECTES-SAPIDUS METALLOTHIONEIN-I THROUGH HETERONUCLEAR (H-1-CD-113) AND HOMONUCLEAR (H-1-H-1) CORRELATION NMR EXPERIMENTS

Metallothionein is a cysteine-rich metal-binding protein whose biosynt hesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, etc., metal salts. The metallothionein from the crab Callinectes sapidus is known to bind six divalent metal ions. Th e Cd-113 NMR spectrum of the Cd-113-substituted protein shows six dist inct Cd-113 resonances spanning a chemical shift range of 620-665 ppm, indicating the presence of six distinct tetrahedrally coordinated div alent metal ion binding sites. Heteronuclear H-1-Cd-113 correlation ex periments revealed all the metal-to-cysteine connectivities present in this protein and the 2X(2(113)Cd)-filtered COSY experiment revealed t hat six cysteines are involved in the ligation of two Cd2+ ions. Addit ionally, following the complete sequential resonance assignment of the H-1 NMR data, the two separate Cd(3)Cys(9) metal binding clusters can be shown to reside in two distinct protein domains.