Establishment of two distinct protein domains in blue crab Callinectes sapidus metallothionein‐I through heteronuclear (¹H¹¹³Cd) and homonuclear ¹H¹H) correlation NMR experiments

Metallothionein is a cysteine‐rich metal‐binding protein whose biosynthesis 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. The ¹¹³Cd NMR spectrum of the ¹¹³Cdsubstituted protein shows six distinct ¹¹³Cd resonances spanning a chemical shift range of 620–665 ppm, indicating the presence of six distinct tetrahedrally coordinated divalent metal ion binding sites. Heteronuclear ¹H¹¹³Cd correlation experiments revealed all the metal‐to‐cysteine connectivities present in this protein and the 2X(2¹¹³Cd)‐;filtered COSY experiment revealed that six cysteines are involved in the ligation of two Cd²⁺ ions. Additionally, following the complete sequential resonance assignment of the ¹H NMR data, the two separate Cd₃Cys₉ metal binding clusters can be shown to reside in two distinct protein domains.