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首页> 外文期刊>Biochemistry >Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular cdc42 signaling.
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Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular cdc42 signaling.

机译：基于遗传编码的荧光共振能量转移的细胞cdc42信号传感器的合理设计。

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摘要

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/orCdc42-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.
机译：Rho GTPase信号通路的时间和空间控制是理解产生复杂细胞运动的分子机制的中心问题。 Rho蛋白Cdc42在其下游效应子Wiskott-Aldrich综合征蛋白（WASP）上诱导了显着的构象变化。基于这种构象变化，我们为活性Cdc42和Cdc42鸟嘌呤核苷酸交换因子（GEF）创建了一系列单分子传感器，这些传感器利用了青色和黄色荧光蛋白之间的荧光共振能量转移（FRET）。在体外，Cdc42传感器在结合活性Cdc42时产生高达3.2倍的FRET发射比变化。在将GDP换成GTP时，GEF传感器的FRET变化高达1.7倍。 GEF传感器对核苷酸的GEF催化的核苷酸交换速率与野生型Cdc42的区别不明显，但GAP催化的核苷酸水解速度减慢了约16倍。在体内，两个传感器均能忠实地报告Cdc42和/或Cdc42-GEF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，这些工具可用于对生命系统中生物学和医学上重要的分子的活性进行成像。

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