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

Peter Kuan Yeu Pan, Z. F. Zheng, P. C. Lyu, P C Huang

Research output: Contribution to journalArticle

Abstract

A novel peptide, the backward reading sequence of human metaliothionein- 2 α domain, was synthesized and its chemical and spectroscopic properties analyzed. This folded retro-α domain was able to bind Cd(II) in identical stoichiometries with the chemically Synthesized α domain of metallothionein- 2. Nearly identical to the α domain, Cd-binding retro-α 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 α domain and retro-α 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-α 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 α 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 α domain and retro-α domain showed that the reversal direction of the domain sequence backbone significantly affects the formation of structure even when it is foldable.

Original languageEnglish (US)
Pages (from-to)33-39
Number of pages7
JournalEuropean Journal of Biochemistry
Volume266
Issue number1
DOIs
StatePublished - Nov 15 1999

Fingerprint

Metallothionein
Metals
Titration
Dithionitrobenzoic Acid
Sulfhydryl Reagents
Acidification
Cadmium
Stoichiometry
Cysteine
Charge transfer
Reading
Absorption spectra
Amino Acid Sequence
Proteins
Amino Acids
Peptides

Keywords

  • Folding nucleus
  • Metal-binding protein
  • Metallothiohein
  • Protein design
  • Retro-protein

ASJC Scopus subject areas

  • Biochemistry

Cite this

Why reversing the sequence of the α domain of human metallothionein-2 does not change its metal-binding and folding characteristics. / Pan, Peter Kuan Yeu; Zheng, Z. F.; Lyu, P. C.; Huang, P C.

In: European Journal of Biochemistry, Vol. 266, No. 1, 15.11.1999, p. 33-39.

Research output: Contribution to journalArticle

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