Seth Abhinav, Otomo Takanori, Yin Helen L, Rosen Michael K
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Biochemistry. 2003 Apr 15;42(14):3997-4008. doi: 10.1021/bi026881z.
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.
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在其下游效应物威斯科特-奥尔德里奇综合征蛋白（WASP）中诱导显著的构象变化。基于这种构象变化，我们创建了一系列用于活性Cdc42和Cdc42鸟嘌呤核苷酸交换因子（GEF）的单分子传感器，它们利用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发射率变化。GEF传感器在GDP与GTP交换时产生高达1.7倍的FRET变化。GEF传感器的GEF催化核苷酸交换速率与野生型Cdc42的速率无法区分，但GAP催化的核苷酸水解速度减慢了约16倍。在体内，两种传感器都忠实地报告Cdc42和/或Cdc42-GEF活性。这些结果证实成功创建了合理设计和基因编码的工具，可用于对活系统中生物学和医学上重要分子的活性进行成像。
