New biarsenical Ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications
New biarsenical Ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications
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DOI：
10.1021/ja017687n
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发表时间：
2002-05-29
影响因子：
15
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Adams, SR;Campbell, RE;Tsien, RY
我们最近引入了一种方法（Griffin，B.一、亚当斯，S.的R.;钱河y. Science 1998，281，269 - 272和Griffin，B.一、亚当斯，S.的R.; Jones，J.;钱河y. methods enzymol. 2000，327，565 - 578）用于活细胞中重组蛋白的位点特异性荧光标记。序列Cys-Cys-Xaa-Xaa-Cys-Cys，其中Xaa是非半胱氨酸氨基酸，被基因融合或插入蛋白质内，在那里它可以被具有两个As（III）取代基的膜渗透性荧光素衍生物RASH特异性识别，RASH仅在砷与半胱氨酸巯基结合后才发出荧光。我们现在报告FIAsH与模型四半胱氨酸肽结合的动力学和解离常数（类似于10（-11）M）。体外亲和力和活细胞中的检测限用Xaa-Xaa = Pro-Gly优化，表明优选的肽构象是发夹而不是先前提出的α-螺旋。已经合成了许多RASH的类似物，包括ReAsH，一种在590 nm处可激发并发出红色荧光的试卤灵衍生物。类似的双砷化合物能够进行亲和层析、荧光各向异性测量和四半胱氨酸标记蛋白质的电子显微镜定位。
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327,565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, RASH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants (similar to10(-11) M) for FIAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of RASH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.