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

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