Construction of an additional metal-binding site in human metallothionein-2

Mitsutoshi Toyama, Mariko Sasaki, Noriaki Hirayama, Yoshikatsu Murooka, Mitsuo Yamashita

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

We have constructed a new metal-binding site in the human metallothionein-2 (hMT-2), using the protein as a scaffold to investigate the structure and function of metal-binding. Potential metal-binding sites were designed within hMT-2 on the basis of structures generated by homology modeling. Amino acid residues D11, C13, C26 and S28 in the β-domain of hMT-2 (hMT-2β) were found, by computer search, to form a potential tetrahedral Cys4 metal-binding site. Six mutant proteins were constructed with the following amino acid substitutions: D11C, S28C and D11C/S28C in hMT-2 and the same mutations in hMT-2β, respectively. These single-mutant and double-mutant proteins bound one gram atom of cadmium or zinc ions per gram molecule of protein more than the corresponding wild-type proteins. The circular dichroism spectra suggested that the structures of the single-mutant proteins that bound Cd or Zn were similar to that of the D11C/S28C double-mutant proteins. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins. The stability with changes in pH of all the mutant proteins was higher than that of the wild-type proteins, and that of the double-mutant D11C/S28C protein was highest. Consequently, it appears that we were able to create novel proteins that bound metal ions at high density and with high affinity.

Original languageEnglish
Pages (from-to)354-360
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume101
Issue number4
DOIs
Publication statusPublished - 2006 Apr
Externally publishedYes

Fingerprint

Metallothionein
Binding sites
Mutant Proteins
Metals
Binding Sites
Proteins
Amino Acids
Cadmium
Titration
Scaffolds
Amino acids
Metal ions
Zinc
Substitution reactions
Ions
Atoms
Molecules
Dichroism
Scaffolds (biology)

Keywords

  • homology modeling
  • metal-thiolate
  • metallothionein
  • molecular orbital calculation
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering

Cite this

Construction of an additional metal-binding site in human metallothionein-2. / Toyama, Mitsutoshi; Sasaki, Mariko; Hirayama, Noriaki; Murooka, Yoshikatsu; Yamashita, Mitsuo.

In: Journal of Bioscience and Bioengineering, Vol. 101, No. 4, 04.2006, p. 354-360.

Research output: Contribution to journalArticle

Toyama, Mitsutoshi ; Sasaki, Mariko ; Hirayama, Noriaki ; Murooka, Yoshikatsu ; Yamashita, Mitsuo. / Construction of an additional metal-binding site in human metallothionein-2. In: Journal of Bioscience and Bioengineering. 2006 ; Vol. 101, No. 4. pp. 354-360.
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