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 (R0) 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. R0 can be experimentally determined using flexible peptide linkers of known lengths to separate the two chromophores. Knowledge of the R0 helps to inform on the choice of BRET system. For example, we have previously shown that BRET2 exhibits the largest R0 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 R0 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 BRET6, a red-shifted BRET2-like system. This study revealed R0 values of 6.15 nm and 6.94 nm for NanoBRET using TMR or NCT as acceptor ligands, respectively. R0 was 5.43 nm for NanoLuc-mCherry, 5.59 nm for NanoLuc-Venus and 5.47 nm for BRET6. 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 BRET2 as an analysis tool for large macromolecular complexes.