Binuclear Ni II -DpaTyr complex as a high affinity probe for an oligo-aspartate tag tethered to proteins

Akio Ojida, Sho Hei Fujishima, Kei Honda, Hiroshi Nonaka, Shohei Uchinomiya, Itaru Hamachi

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni II -DpaTyr (DpaTyr = bis((dipicolylamino)methyl)-tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M = Zn II , Ni II , Mn II , Cu II , Cd II , Co III , and Fe III ), we have found that Ni II -DpaTyr (1-2Ni II ) displays a strongbinding affinity (apparent binding constant: K app ≈10 5 M -1 ) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni II in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni II -and Zn II - DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni II -DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K app 10 9 M -1 ) was achieved between the Ni II -DpaTyr dimer 4-4Ni II and the D3 x 2 tag peptide (DDDNGDDD). This affinity is ≈fold stronger than that observed in the binding pair of the Zn II -DpaTyr (4-4Zn II ) and the D4x2 tag (DDDDGDDDD), a useful tagprobe pair previously reported by us. The recognition pair of the Ni II -DpaTyr probe and the D3 x 2 tag can also work effectively on a protein surface, that is, 4-4Ni II is strongly bound to the FKBP12 protein tethered with the D3 x 2 tag (DDDNGDDD) with a large K app value of 5 x 10 8 m -1 . Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tagfused (β-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.

Original languageEnglish
Pages (from-to)877-886
Number of pages10
JournalChemistry - An Asian Journal
Volume5
Issue number4
DOIs
Publication statusPublished - Apr 1 2010
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Organic Chemistry

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