Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Bioorg Med Chem Lett. 2011 Sep 1;21(17):5058-61. doi: 10.1016/j.bmcl.2011.04.051. Epub 2011 Apr 20.
Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.
CDC42，Rho GTPase 家族的一员，是细胞迁移过程中肌动蛋白细胞骨架的基本调节因子。为了产生一个用于检测 Cdc42 激活的传感器，我们采用了多管齐下的方法，利用半胱氨酸标记和表达蛋白连接，将环境敏感荧光团 4-N,N-二甲基氨基-1,8-萘酰亚胺（4-DMN）整合到 WAVE 蛋白的 GTP 结合域中。这些构建体仅与活性、GTP 结合的 Cdc42 结合，以产生荧光信号。使用一组五个传感器类似物的研究表明，与与无活性 GDP 结合形式的蛋白孵育相比，在存在激活的 Cdc42 时，该传感器的衍生物的荧光强度增加了 32 倍。我们证明，该传感器可用于在连续荧光测定中监测 Cdc42 核苷酸交换和 GTP 酶活性。
