Chemistry - An Asian Journal p. 877 - 886 (2010)
Update date:2022-07-30
Topics:
Ojida, Akio
Fujishima, Sho-Hei
Honda, Kei
Nonaka, Hiroshi
Uchinomiya, Sho-Hei
Hamachi, Itaru
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 NiII-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, NiII, MnII, CuII, Cd II, CoIII, and FeIII), we have found that NiII-DpaTyr (1-2NiII) displays a strongbinding affinity (apparent binding constant: Kapp≈105M-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 1-2NiII in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for NiII-and ZnII- 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 (Kapp 10 9M-1) was achieved between the NiII-DpaTyr dimer 4-4NiII 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 (4-4ZnII) 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, 4-4NiII is strongly bound to the FKBP12 protein tethered with the D3 x 2 tag (DDDNGDDD) with a large Kapp value of 5 x 108 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.
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