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

21 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
Externally publishedYes

Keywords

  • Ascidian
  • Metal-binding protein
  • Vanadium

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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