Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10⁻⁹ to 10⁻⁶ M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.

中文翻译：

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针对 ATP7B 金属结合域的纳米抗体作为研究细胞内铜转运的工具

纳米抗体是基因工程单域抗体，源自美洲驼和骆驼中发现的不寻常的仅重链抗体。纳米抗体的小尺寸和灵活的选择方案使其成为蛋白质生物化学和细胞生物学独特的多功能工具。我们开发了一组针对人铜转运蛋白 ATP7B 金属结合域的纳米抗体，ATP7B 是一种多域膜蛋白，对酶活性和细胞内定位具有复杂的调节作用。为了能够使用纳米抗体作为研究细胞中铜转运的工具，我们通过等温滴定量热法和核磁共振来表征它们的结合位点和亲和力。我们已经鉴定出针对 ATP7B 前四个金属结合域中每一个的纳米抗体，其亲和力范围较宽，解离常数低于 10 -9^至10 ^-6 M 即可证明。我们在测试的纳米抗体中发现了抑制性和激活性纳米抗体。纳米抗体的多种特性使该面板可用于 ATP7B 的结构研究、蛋白质的免疫亲和纯化、细胞中活性的调节、蛋白质动力学研究，以及作为铜伴侣 ATOX1（ATP7B 的天然相互作用伴侣）的模拟物。