Vuojola Johanna, Lamminmäki Urpo, Soukka Tero
Department of Biotechnology, University of Turku, FI-20520 Turku, Finland.
Anal Chem. 2009 Jun 15;81(12):5033-8. doi: 10.1021/ac9005793.
Forster resonance energy transfer (FRET) is a powerful tool in studying biomolecular interactions. Intrinsically fluorescent lanthanide chelates are increasingly used as FRET donors due to their long emission lifetime that enables the use of time resolution. Fluorescent proteins, on the other hand, owe their popularity to the intrinsic luminescent properties, facilitating their use as fusion proteins. In this investigation, two energy transfer pairs, terbium(III) chelate with green fluorescent protein (GFP) and europium(III) chelate with yellow fluorescent protein (YFP), were studied by expressing the fluorescent protein acceptor as a fusion protein together with Rab21 GTPase. GTP-conjugated lanthanide chelates were used as donor conjugates. In contrast to conventional FRET observed with the Tb(3+)-GFP pair, a phenomenon called nonoverlapping FRET (nFRET) was observed with the Eu(3+)-YFP pair. In nFRET, the sensitized emission of the acceptor was measured at shorter wavelength than where the emission of the donor was observed. Regardless of the lower signal levels, nFRET resulted in a substantially higher signal-to-background ratio. Conventional FRET from sensitized acceptor yielded a single apparent fluorescence emission lifetime, while with nFRET two lifetimes were observed. The lanthanide chelates together with fluorescent proteins enable a straightforward and sensitive assay technology in nFRET applications.
福斯特共振能量转移（FRET）是研究生物分子相互作用的有力工具。由于其长发射寿命能够实现时间分辨，本征荧光镧系螯合物越来越多地被用作FRET供体。另一方面，荧光蛋白因其本征发光特性而广受欢迎，便于用作融合蛋白。在本研究中，通过将荧光蛋白受体与Rab21 GTP酶一起表达为融合蛋白，研究了两个能量转移对，即铽（III）螯合物与绿色荧光蛋白（GFP）以及铕（III）螯合物与黄色荧光蛋白（YFP）。GTP共轭镧系螯合物用作供体共轭物。与用Tb(3+)-GFP对观察到的传统FRET不同，用Eu(3+)-YFP对观察到一种称为非重叠FRET（nFRET）的现象。在nFRET中，受体的敏化发射在比观察到供体发射的波长更短的波长处测量。尽管信号水平较低，但nFRET导致的信背比显著更高。来自敏化受体的传统FRET产生单一的表观荧光发射寿命，而在nFRET中观察到两个寿命。镧系螯合物与荧光蛋白一起在nFRET应用中实现了一种直接且灵敏的检测技术。
