Fluorescent Cassettes for Monitoring Three-Component Interactions in Vitro and in Living Cells

Two water-soluble “through-bond energy transfer cassettes” (TBET-cassettes) were prepared. They have good extinction coefficients at the donor part and transfer energy to the acceptor parts with good “overall quantum yields” (0.30 and 0.24 in pH 7.4 phosphate buffer). Fluorescence resonance energy transfer from one protein functionalized with an appropriate donor can be used to excite the TBET-cassettes on different proteins to probe protein−protein interactions under conditions that would not be possible for single-dye acceptor systems.