Anomalous photophysical behaviors attributed to excitonic coupling in fluorescent proteins

Fluorescent proteins (FPs) comprise hundreds of different genetically encoded biosensors. Anomalous FP photo-physics, consistent with excitonic coupling, i.e., delocalized excitation, has been previously reported. Since delocalized excitation can potentially alter fluorescence lifetimes, intensities, and spectra at long distances, its impact needs to be evaluated for the proper design and interpretation of biosensor experiments, as well as for the development of genetically encoded excitonic materials. In addition, it is unclear if excitonic coupling is a shared trait of all β-barrel FPs, nor are the distances requirements for FP excitonic coupling known. To address these questions, we engineered FP constructs having either one or two functional chromophores. We found red shifts in absorption, circular dichroism Davydov splitting, and shorter fluorescence lifetimes in evolutionarily divergent FP tandem dimers having two chromophores, supporting the existence of excitonic coupling. Photon antibunching statistics indicated that tandem dimers with two chromophores emit as a single quantum unit. Sub-Poissonian photon statistics was observed even with 20-nm Venus-Venus chromophore separation, twice the limit of Förster's resonance energy transfer, but not at 60-nm. Our findings support the hypothesis that the conserved β-barrel structure is the common structural attribute associated with allowing fluorescent protein exciton delocalization under physiological conditions.