Binuclear Ni II ‐DpaTyr Complex as a High Affinity Probe for an Oligo‐Aspartate Tag Tethered to Proteins

• Published in: Chemistry, an Asian journal an Asian journal, 2010-04, Vol.5 (4), p.877-886
• Main Authors: Ojida, Akio, Fujishima, Sho‐hei, Honda, Kei, Nonaka, Hiroshi, Uchinomiya, Sho‐hei, Hamachi, Itaru
• Format: Article
• Language: English
• ISSN: 1861-4728; 1861-471X
• DOI: 10.1002/asia.200900362

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 ( 1‐2Ni2+ ) displays a strong‐binding affinity (apparent binding constant: K app ≈10 5   M −1 ) for an oligo‐aspartate peptide under neutral aqueous conditions (50 m M HEPES, 100 m M NaCl, pH 7.2). Detailed isothermal‐titration calorimetry (ITC) studies reveal that the tri‐aspartate D3‐tag (DDD) is an optimal sequence recognized by 1‐2Ni2+ 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 (D3×2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high‐binding affinity ( K app =2×10 9   M −1 ) was achieved between the Ni II ‐DpaTyr dimer 4‐4Ni2+ and the D3×2 tag peptide (DDDNGDDD). This affinity is ≈100‐fold stronger than that observed in the binding pair of the Zn II ‐DpaTyr ( 4‐4Zn2+ ) and the D4×2 tag (DDDDGDDDD), a useful tag‐probe pair previously reported by us. The recognition pair of the Ni II ‐DpaTyr probe and the D3×2 tag can also work effectively on a protein surface, that is, 4‐4Ni2+ is strongly bound to the FKBP12 protein tethered with the D3×2 tag (DDDNGDDD) with a large K app value of 5×10 8   M −1 . Taking advantage of the strong‐binding affinity, this pair was successfully applied to the selective inactivation of the tag‐fused β‐galactosidase by using the chromophore‐assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.