Association of fluorescent protein pairs and it's significant impact on fluorescence and energy transfer

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Forster resonance energy transfer (FRET). We assessed the impact of FP association by predicting dimerisation sites in silico and stabilising the dimers by bioorthogonal covalent linkages. In each tested case dimerisation changed inherent fluorescence, including FRET. GFP homodimers demonstrated synergistic behaviour with the dimer being brighter than the sum of the monomers. The homodimer structure revealed the chromophores were close with favourable transition dipole alignments and a highly solvated interface. Heterodimerisation (GFP with Venus) resulted in a complex with ~87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency was observed when the wild-type FPs were fused to a naturally occurring protein-protein interface system. GFP complexation with mCherry resulted in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.