Authors
Tuan Nguyen, Henry L Puhl, Eefei Chen, Kirk Hines, Chanchal Rani, Paul S Blank, Benedetta Carlotti, Youngchan Kim, Theodore Goodson, Steven S Vogel
Published in
Biophysical journal. Oct 18, 2025. Epub Oct 18, 2025.
Abstract
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 is 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 (TDs) having two chromophores, supporting the existence of excitonic coupling. Photon antibunching statistics indicated that TDs 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 (FRET), 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.
PMID:
41109955
Bibliographic data and abstract were imported from PubMed on 19 Oct 2025.
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