Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling.
Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling.
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基于基因编码荧光共振能量转移的细胞 Cdc42 信号传导传感器的合理设计。
DOI：
10.1021/bi026881z
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Rosen,MichaelK
中科院分区：
文献类型：
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作者：
Seth,Abhinav;Otomo,Takanori;Yin,HelenL;Rosen,MichaelK
Rho GTPase信号通路的时空控制是理解产生复杂细胞运动的分子机制的核心问题。Rho蛋白Cdc42诱导其下游效应蛋白Wiskott - Aldrich综合征蛋白（WASP）发生显著的构象变化。在这种构象变化的基础上，我们创建了一系列单分子传感器，用于活性Cdc42和Cdc42鸟嘌呤核苷酸交换因子（GEFs），利用荧光共振能量转移（FRET）在青色和黄色荧光蛋白之间。在体外，Cdc42传感器结合活性Cdc42后产生高达3.2倍的FRET发射比变化。GEF传感器产生高达1.7倍的变化，FRET在交换GDP为GTP。GEF催化的GEF传感器的核苷酸交换速率与野生型Cdc42几乎没有区别，但gap催化的核苷酸水解速度减慢了大约16倍。在体内，这两种传感器都忠实地报告Cdc42和/或Cdc42- gef的活性。这些结果建立了合理设计和遗传编码工具的成功创造，可用于成像生物和医学上重要分子在生命系统中的活动。
The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott−Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.