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 strong-binding affinity (apparent binding constant: K(app) approximately 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)=2x10(9) M(-1)) was achieved between the Ni(II)-DpaTyr dimer 4-4Ni(II) and the D3x2 tag peptide (DDDNGDDD). This affinity is approximately 100-fold stronger than that observed in the binding pair of the Zn(II)-DpaTyr (4-4Zn(II)) and the D4x2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni(II)-DpaTyr probe and the D3x2 tag can also work effectively on a protein surface, that is, 4-4Ni(II) is strongly bound to the FKBP12 protein tethered with the D3x2 tag (DDDNGDDD) with a large K(app) value of 5x10(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.
Ojida
Akio
A
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, Japan.
Fujishima
Sho-Hei
SH
Honda
Kei
K
Nonaka
Hiroshi
H
Uchinomiya
Sho-Hei
SH
Hamachi
Itaru
I
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D002151:Calorimetry; D056831:Coordination Complexes / Q000138:chemical synthesis / Q000737:chemistry*; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry; D009532:Nickel / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry
D056831:Coordination Complexes; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011506:Proteins; D001224:Aspartic Acid; D009532:Nickel; D001616:beta-Galactosidase; D022061:Tacrolimus Binding Protein 1A