Experimental Determination of the Forster Distance for Two Commonly Used Bioluminescent Resonance Energy Transfer Pairs

Forster resonance energy transfer (RET) is the nonradiative transfer of energy from a donor to an acceptor fluorophore. The Forster distance (R(0)), being the fluorophore separation corresponding to 50% of the maximum RET efficiency (E(RET)) is a critical parameter for optimization of RET biosensors. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Forster distance. Here, for the first time, we experimentally determine the Forster distance for BRET(1), R(0) = 4.4 nm, and for BRET(2), R(0) = 7.5 nm. The latter is the largest reported value for a genetically encoded RET pair.