Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.
Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.
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DOI：
10.1038/srep19211
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发表时间：
2016-01-21
影响因子：
4.6
通讯作者：
Rothbauer U
中科院分区：
文献类型：
--
作者：
Braun MB;Traenkle B;Koch PA;Emele F;Weiss F;Poetz O;Stehle T;Rothbauer U
纳米抗体是许多生物分析和生物技术应用的非常有价值的工具。在这里，我们报告了一种纳米抗体的表征，该纳米抗体以非凡的亲和力结合短肽表位。结构分析揭示了一种不寻常的结合模式，其中延伸的肽成为纳米抗体中β折叠结构的一部分。这种相互作用依赖于由少量特异性决定侧链接触增强的序列独立性骨架相互作用。一旦结合，肽就被两个纳米抗体侧链固定，这两个纳米抗体侧链以头锁的方式夹住肽。利用这种不寻常的结合模式，我们产生了一种新的纳米抗体衍生的捕获和检测系统。基质偶联的纳米抗体使得能够从原核和真核表达系统中快速有效地分离表位标记的蛋白质。此外，荧光标记的纳米抗体使不同细胞区室中的亚细胞结构可视化。该系统的高亲和力结合和可修饰的肽标签使其成为将联合收割机生化分析与显微镜研究相结合的通用和强大的工具。
Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.