title: 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.
authors.affiliation: Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, China.
authors.forename: P K
authors.initials: PK
authors.lastname: Pan
authors.forename: Z F
authors.initials: ZF
authors.lastname: Zheng
authors.forename: P C
authors.initials: PC
authors.lastname: Lyu
authors.forename: P C
authors.initials: PC
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004136:Dinitrobenzenes / Q000378:metabolism*; D006801:Humans; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D004136:Dinitrobenzenes; C491053:MT2A protein, human; C491055:Mt2A protein, rat; D010455:Peptides; C004203:cadmium-binding protein; C116509:cadmium-metallothionein complex; D002104:Cadmium; D008668:Metallothionein; C010936:2,4-dinitrothiocyanatobenzene; D003545:Cysteine