Robust Red FRET Sensors Using Self-Associating Fluorescent Domains

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• 作者：Laurens H. Lindenburg ; Anne M. Hessels ; Eduard H. T. M. Ebberink ; Remco Arts ; Maarten Merkx
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：October 18, 2013
• 年：2013
• 卷：8
• 期：10
• 页码：2133-2139
• 全文大小：460K
• 年卷期：v.8,no.10(October 18, 2013)
• ISSN：1554-8937

文摘

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn²⁺ sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn²⁺ FRET sensors to image Zn²⁺ over a broad concentration range in the same cellular compartment.