Abstract
Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Fo ̈rster’s resonance energy transfer (FRET) measurements, FP-tagged proteins can be used tostudy protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombicdipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and inco-herently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy isdistributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The inter-pretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weaklycoupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typi-cally associated with strong excitonic coupling, is limited by the FPb-barrel structure. Furthermore, physiological temperaturespromote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasingtimes that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolutionhas shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitoniccoupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by moni-toring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause achange in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopyrevealed Davydov splitting when the yellow FP VenusA206dimerizes, and a novel approach combining photon antibunching andfluorescence correlation spectroscopy was used to confirm that the two fluorophores in a VenusA206homodimer behave as asingle-photon emitter. We conclude that excitonic coupling between VenusA206fluorophores is possible at physiologicaltemperatures