Resonance energy transfer from lanthanide chelates to overlapping and nonoverlapping fluorescent protein acceptors

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.