Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, Japan.
Chem Asian J. 2010 Apr 1;5(4):877-86. 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 (1-2Ni(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.
一对短肽标签和小分子探针的互补识别对蛋白质的检测、处理和纯化等具有重要意义。在本文中，我们报告了双核 Ni(II)-DpaTyr（DpaTyr=双（（二吡啶基氨基）甲基）酪氨酸）配合物作为与蛋白质相连的寡天冬氨酸标签的强结合探针。在各种双核金属配合物 M-DpaTyr（M=Zn(II)，Ni(II)，Mn(II)，Cu(II)，Cd(II)，Co(III)和 Fe(III)）中，我们发现 Ni(II)-DpaTyr（1-2Ni(II)）在中性水条件下（50 mM HEPES，100 mM NaCl，pH 7.2）对寡天冬氨酸肽具有很强的结合亲和力（表观结合常数：K(app)约为 10(5) M(-1))。详细的等温滴定量热法（ITC）研究表明，三天冬氨酸 D3 标签（DDD）是 1-2Ni(II)以 1:1 结合比例识别的最佳序列。另一方面，除了 Ni(II)-和 Zn(II)-DpaTyr 之外的其他 DpaTyr 金属配合物对寡天冬氨酸肽几乎没有结合亲和力。基于它们之间的多价配位相互作用，双核 Ni(II)-DpaTyr 的二聚体与重复的 D3 标签肽（例如 DDDXXDDD）之间的结合对大大增强了结合亲和力。值得注意的是，Ni(II)-DpaTyr 二聚体 4-4Ni(II)与 D3x2 标签肽（DDDNGDDD）之间实现了非常高的结合亲和力（K(app)=2x10(9) M(-1))。这种亲和力比我们之前报道的有用的 Zn(II)-DpaTyr（4-4Zn(II)）和 D4x2 标签（DDDDGDDDD）之间的结合对强 100 倍。Ni(II)-DpaTyr 探针和 D3x2 标签的识别对也可以在蛋白质表面有效发挥作用，即 4-4Ni(II)与 D3x2 标签（DDDNGDDD）结合的 FKBP12 蛋白具有很大的 K(app)值（5x10(8) M(-1))。利用强结合亲和力，该对成功应用于在粗条件下（例如细胞裂解物）通过发色团辅助光失活（CALI）技术选择性失活标记融合的β-半乳糖苷酶。
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