A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure
A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure
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DOI：
10.1073/pnas.0611713104
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发表时间：
2007-03-06
影响因子：
11.1
通讯作者：
Tsien, Roger Y.
中科院分区：
文献类型：
--
作者：
Hauser, Christina T.;Tsien, Roger Y.
蛋白质在体内的位点特异性荧光标记仍然是活细胞中成像复杂过程的最强大的技术之一。虽然许多颜色的荧光蛋白是用于跟踪细胞中融合蛋白的表达和定位的有用工具，但是这些相对较大的标签（> 220 aa）可以干扰蛋白质折叠、运输和功能。小得多的遗传编码结构域（< 15 aa）提供互补优势。我们介绍了一种小的荧光螯合剂，其膜不渗透复合物与无毒的锌离子紧密结合，但可逆的表面暴露的蛋白质上的六聚组氨酸（嘶嘶声）图案。这种活细胞标签有助于解决目前的争议，关于外化的基质相互作用分子STIM 1后，从内质网中的钙耗尽。尽管N-末端荧光蛋白融合干扰STIM 1的表面暴露，但短His（6）标签可接近染料或抗体，表明外部化。
Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (> 220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (< 15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (Hiss) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His(6) tags are accessible to the dye or antibodies, demonstrating externalization.