Why reversing the sequence of the alpha domain of human metallothionein-2 does not change its metal-binding and folding characteristics.

Abstract

A novel peptide, the backward reading sequence of human metallothionein-2 alpha domain, was synthesized and its chemical and spectroscopic properties analyzed. This folded retro-alpha domain was able to bind Cd(II) in identical stoichiometries with the chemically synthesized alpha domain of metallothionein-2. Nearly identical to the alpha domain, Cd-binding retro-alpha domain showed a characteristic ultraviolet absorption spectrum with a shoulder at 245-250 nm (due to cadmium-thiolate charge transfer), and the absorption shoulder was abolished by acidification [suggesting mercaptide bonding between Cd(II) and the cysteine residues]. Similar metal-binding capabilities between alpha domain and retro-alpha domain were observed also by pH titration and in the reaction with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid). A two-state cooperativity of the metal-cluster formation was observed spectroscopically in the titration of the retro-alpha domain, indicating that the retro-protein is foldable. In contrast to other proteins, our results indicate that the reversion of the amino acid sequence for the alpha domain does not change its foldability and metal-binding capacity, suggesting that the order of its sequence is not critical to the formation of a critical metal-tetrathiolate nucleus. However, CD spectra of the Cd-binding alpha domain and retro-alpha domain showed that the reversal direction of the domain sequence backbone significantly affects the formation of structure even when it is foldable.