Experimental determination of the bioluminescence resonance energy transfer (BRET) Förster distances of NanoBRET and red-shifted BRET pairs

Bioluminescence Resonance Energy Transfer (BRET) is widely applied to study protein-protein interactions, as well as increasingly to monitor both ligand binding and molecular rearrangements. The Förster distance (R(0)) describes the physical distance between the two chromophores at which 50% of the maximal energy transfer occurs and it depends on the choice of RET components. R(0) can be experimentally determined using flexible peptide linkers of known lengths to separate the two chromophores. Knowledge of the R(0) helps to inform on the choice of BRET system. For example, we have previously shown that BRET(2) exhibits the largest R(0) to date for any genetically encoded RET pair, which may be advantageous for investigating large macromolecular complexes if its issues of low and fast-decaying bioluminescence signal can be accommodated. In this study we have determined R(0) for a range of bright and red-shifted BRET pairs, including NanoBRET with tetramethylrhodamine (TMR), non-chloro TOM (NCT), mCherry or Venus as acceptor, and BRET(6), a red-shifted BRET(2)-like system. This study revealed R(0) values of 6.15 nm and 6.94 nm for NanoBRET using TMR or NCT as acceptor ligands, respectively. R(0) was 5.43 nm for NanoLuc-mCherry, 5.59 nm for NanoLuc-Venus and 5.47 nm for BRET(6). This extends the palette of available BRET Förster distances, to give researchers a better-informed choice when considering BRET systems and points towards NanoBRET with NCT as a good alternative to BRET(2) as an analysis tool for large macromolecular complexes.