The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the 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. Abstract Figure