Ecole Normale Supérieure, Département de Physique, Laboratoire de Physique Statistique UMR CNRS-ENS 8550, 24 rue Lhomond, 75231 Paris, France.
Chembiochem. 2010 Mar 22;11(5):653-63. doi: 10.1002/cbic.201000008.
We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.
我们已经开发出一种非侵入性的光学方法，可用于快速控制活体斑马鱼胚胎中的蛋白质活性。该方法依赖于将与细胞质伴侣复合物结合的蛋白质释放出来，该蛋白质与经过修饰的雌激素受体配体结合域融合在一起，而这一过程是通过局部光激活非内源性的被笼闭的诱导剂来实现的。分子动力学模拟用于设计环芬-OH，这是一种对 4-羟基他莫昔芬（ER(T2)）具有特异性的受体的光稳定诱导剂。环芬-OH 可以很容易地通过两步合成，产率良好。在亚毫摩尔浓度下，它可以激活与 ER(T2)受体融合的蛋白质。这在培养细胞和斑马鱼胚胎中通过发射特性和经过适当设计的荧光蛋白的亚细胞定位得到了证明。环芬-OH 可以与各种光不稳定的保护基团成功地笼闭。一种特定的被笼闭的化合物能够有效地用光诱导荧光蛋白的核转位，无论是全局（使用 365nmUV 照射）还是局部（使用聚焦的 UV 激光或 750nm 的双光子照射）。这种用于蛋白质活性光控制的方法可以更广泛地用于研究重要的生理过程（例如在胚胎发生、器官再生和癌症发生过程中），具有高时空分辨率。
