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Relevant academic research and scientific papers

Design of coordination interaction of Zn(II) complex with oligo-aspartate peptide to afford a high-affinity tag-probe pair

A complementary recognition pair consisting of a genetically encodable peptide tag and a small molecular probe isa powerful tool to specifically label and manipulate a protein ofinterest under biological conditions. In this study, we report the redesign of a tag-probe pair comprising an oligo-aspartate peptide tag (such as DDDD) and a binuclear zinc complex. Isothermal-titration calorimetry screening of binding between the series of peptides and zinc complexes revealed that the binding affinity was largely influenced by subtle changes of the ligand structure of the probe. However, the binding was tolerant to differences of the tag peptide sequence. Of those tested, a pair containing a peptide tag (DDAADD) and a binuclear zinc complex possessing 4-chloropyridines (3-2Zn(II)) showed the strongest binding affinity (Ka = 3.88 × 105 M-1), which was about 10-fold larger than the conventional pair of D4-peptide tag (DDDD) and 1-2Zn(II) containing nonsubstituted pyridines (Ka = 3.73 × 104 M-1). The strong binding of this new complementary recognition pair enabled the rapid covalent labeling of a tag-fused maltose binding protein with a fluorescent zinc complex, demonstrating its potential utility in protein analysis.

Binuclear NiII-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 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.