Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

Tatsuya Ueki, Norifumi Kawakami, Masaaki Toshishige, Koichi Matsuo, Kunihiko Gekko, Hitoshi Michibata

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Background: Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. Methods: In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Results: Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. General significance: These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+.

Original languageEnglish
Pages (from-to)1327-1333
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Volume1790
Issue number10
DOIs
Publication statusPublished - 2009 Oct 1
Externally publishedYes

Fingerprint

Vanadium
Mutagenesis
Site-Directed Mutagenesis
Carrier Proteins
Metals
Binding Sites
Urochordata
Disulfides
Mutation
Circular dichroism spectroscopy
Affinity chromatography
Ion chromatography
Metal ions
Circular Dichroism
Affinity Chromatography
Nuclear magnetic resonance
Spectrum Analysis
Magnetic Resonance Spectroscopy
Ions
Proteins

Keywords

  • Ascidian
  • Metal-binding protein
  • Vanadium

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis. / Ueki, Tatsuya; Kawakami, Norifumi; Toshishige, Masaaki; Matsuo, Koichi; Gekko, Kunihiko; Michibata, Hitoshi.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1790, No. 10, 01.10.2009, p. 1327-1333.

Research output: Contribution to journalArticle

Ueki, Tatsuya ; Kawakami, Norifumi ; Toshishige, Masaaki ; Matsuo, Koichi ; Gekko, Kunihiko ; Michibata, Hitoshi. / Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis. In: Biochimica et Biophysica Acta - General Subjects. 2009 ; Vol. 1790, No. 10. pp. 1327-1333.
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