Pham Elizabeth, Truong Kevin
Institute of Biomaterials and Biomedical Engineering and Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2010;591:69-91. doi: 10.1007/978-1-60761-404-3_4.
Many fluorescent probes depend on the fluorescence resonance energy transfer (FRET) between fluorescent protein pairs. The efficiency of energy transfer becomes altered by conformational changes of a fused sensory protein in response to a cellular event. A structure-based approach can be taken to design probes better with improved dynamic ranges by computationally modeling conformational changes and predicting FRET efficiency changes of candidate biosensor constructs. FRET biosensors consist of at least three domains fused together: the donor protein, the sensory domain, and the acceptor protein. To more efficiently subclone fusion proteins containing multiple domains, a cassette-based system can be used. Generating a cassette library of commonly used domains facilitates the rapid subcloning of future fusion biosensor proteins. FRET biosensors can then be used with fluorescence microscopy for real-time monitoring of cellular events within live cells by tracking changes in FRET efficiency. Stimulants can be used to trigger a range of cellular events including Ca(2+) signaling, apoptosis, and subcellular translocations.
许多荧光探针依赖于荧光蛋白对之间的荧光共振能量转移（FRET）。响应细胞事件时，融合的传感蛋白的构象变化会改变能量转移效率。通过对构象变化进行计算建模并预测候选生物传感器构建体的FRET效率变化，可以采用基于结构的方法来更好地设计具有改善动态范围的探针。FRET生物传感器至少由三个融合在一起的结构域组成：供体蛋白、传感结构域和受体蛋白。为了更有效地亚克隆包含多个结构域的融合蛋白，可以使用基于盒式的系统。生成常用结构域的盒式文库有助于未来融合生物传感器蛋白的快速亚克隆。然后，可以将FRET生物传感器与荧光显微镜一起使用，通过跟踪FRET效率的变化来实时监测活细胞内的细胞事件。刺激剂可用于触发一系列细胞事件，包括Ca(2+)信号传导、细胞凋亡和亚细胞易位。
