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

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.