Engineering of weak helper interactions for high-efficiency FRET probes
Engineering of weak helper interactions for high-efficiency FRET probes
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DOI：
10.1038/nmeth.2625
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发表时间：
2013-10-01
期刊：
影响因子：
48
通讯作者：
Serrano, Luis
中科院分区：
文献类型：
--
作者：
Gruenberg, Raik;Burnier, Julia V.;Serrano, Luis
基于荧光共振能量转移（FRET）的蛋白质相互作用的检测受到FRET允许距离的非常窄的范围的限制。我们展示了两种不同的策略，用于合理设计弱辅助相互作用，共招募供体和受体荧光团，用于更稳健地检测双分子FRET：（i）静电驱动的遭遇复合物的计算机设计和（ii）基于WW或SH 3结构域的可调结构域-肽相互作用模块的融合。我们测试了用于优化（m）Citrine和mCherry之间的FRET的每种策略，它们不天然相互作用。这两种方法都产生了相当的和大的FRET效率增加很少或没有背景。辅助相互作用模块可以融合到任何一对荧光蛋白，我们发现，可以增强mTFP 1和mCherry之间以及mTurquoise 2和mCitrine之间的FRET。我们应用增强型辅助相互作用FRET（hiFRET）探针研究全长H-Ras和Raf 1之间的结合以及Raf 1和B-Raf之间的药物诱导的相互作用。
Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.