Evers Toon H, Appelhof Marieke A M, Meijer E W, Merkx Maarten
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands.
Protein Eng Des Sel. 2008 Aug;21(8):529-36. doi: 10.1093/protein/gzn029. Epub 2008 May 23.
Fluorescent indicators that allow real-time imaging of Zn(II) in living cells are invaluable tools for understanding Zn(II) homeostasis. Genetically encoded sensors based on fluorescence resonance energy transfer between fluorescent protein domains have important advantages over synthetic probes. We discovered that hexahistidine tags have a strong tendency to dimerize upon binding of Zn(II) in solution and we used this principle to develop a new protein-based sensor for Zn(II). Enhanced cyan and yellow fluorescent proteins were connected by long flexible peptide linkers and His-tags were incorporated at both termini of this fusion protein. The resulting sensor CLY9-2His allows the ratiometric fluorescent detection of Zn(II) in the nanomolar range. In addition, CLY9-2His is selective over the physiologically relevant metal ions Fe(II), Mn(II), Ca(II) and Mg(II). Our approach demonstrates the potential of using small peptides as metal-binding ligands in chelating fluorescent protein chimeras.
能够对活细胞中的锌离子（Zn(II)）进行实时成像的荧光指示剂，是理解锌离子动态平衡的宝贵工具。基于荧光蛋白结构域之间荧光共振能量转移的基因编码传感器，相较于合成探针具有重要优势。我们发现，六组氨酸标签在溶液中与锌离子结合后有很强的二聚化倾向，我们利用这一原理开发了一种新型的基于蛋白质的锌离子传感器。增强型青色和黄色荧光蛋白通过长的柔性肽接头连接，组氨酸标签被引入到这种融合蛋白的两端。由此产生的传感器CLY9-2His能够对纳摩尔范围内的锌离子进行比率荧光检测。此外，CLY9-2His对生理相关金属离子亚铁离子（Fe(II)）、锰离子（Mn(II)）、钙离子（Ca(II)）和镁离子（Mg(II)）具有选择性。我们的方法证明了在螯合荧光蛋白嵌合体中使用小肽作为金属结合配体的潜力。
