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 = Ni-II, Mn-II, Cu-II, Cd-II, Co-II, and Fe-III), we have found that Ni-II-DpaTyr (1-2Ni(II)) displays a strong-binding affinity (apparent binding constant: Kapp approximate to 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 Ze(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 (D3 x 2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K-app=2 x 10(9) M-1) was achieved between the Ni-II-DpaTyr dimer 4-4Ni(II) and the D3 x 2tag peptide (DDDNGDDD). This affinity is,approximate to 1.00-fold stronger than that observed in the binding pair of the Zn-II-DpaTyr (4-4Zn(II)) and the D4 x 2 tag (DDDDGDDDD), a useful tag-probe 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 tag-fused beta-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.