Replacement of terminal cysteine with histidine in the metallothionein alpha and beta domains maintains its binding capacity.

To generate novel forms of metal-binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli. The spectroscopic and analytical results showed that the alphaMT (C33H, C36H, C41H, C57H) and betaMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected. In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd-mutated MT forms with 4 and 3 Cd(II), respectively. However, although Cys-to-His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild-type proteins. While C13H, C36H and C57H seem to contain Zn(II)-aggregates that are closely related to those of the wild-type proteins, only C41H and C57H gave rise to Cd(II)-aggregates similar to those of Cd4-alphaMT, where the His residue plays the role of the substituted Cys. Despite the structural implications of the Cys-to-His replacement, the dissociation constants showed no major decrease in the Cd-binding affinity in any of the mutants assayed compared with the wild-type.