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.