VenusA206 Dimers Behave Coherently at Room Temperature

Abstract

Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Fö rster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP b-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP Venus A206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a Venus A206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between Venus A206 fluorophores is possible at physiological temperatures.

Quantum Biology: An Update and Perspective

“…In 2019, further experimental evidence for room temperature coherent molecular interactions between FP fluorophores was obtained by Kim et al The study reported unexpected FP quantum behaviour resulting from excitonic coupling in homodimers of dimeric yellow fluorescent protein Venus A206 under physiological conditions (Figure 11A) [340]. Time-resolved fluorescence anisotropy measurements were used to reveal ultrafast energy transfer (defined as faster than the instrument response function of the anisotropy system, ~146 ps) between FP fluorophores when Venus A206 FPs dimerise (Figure 11B).…”

“…It was therefore thought to be highly unlikely that coherent energy transfer takes place between fluorescent proteins (FPs) as fluorophore proximity is limited by the FP β-barrel structure and, in a warm, wet biological environment, vibrational decoherence would rapidly intervene to obliterate quantum coherent molecular interactions. Nonetheless, evidence indicates that anomalous photophysical properties of FP have been observed at room temperature, which cannot be understood explicitly within the boundaries of classic physics [337][338][339][340][341][342][343][344][345][346]. Surprisingly, recent experimental evidence observing generation of photonic entanglement in enhanced green fluorescent protein (eGFP) [339] and exciton coupling between dimeric yellow fluorescent proteins, Venus, [340] strongly supports the hypothesis that FPs can interact coherently under physiological conditions.…”

“…Nonetheless, evidence indicates that anomalous photophysical properties of FP have been observed at room temperature, which cannot be understood explicitly within the boundaries of classic physics [337][338][339][340][341][342][343][344][345][346]. Surprisingly, recent experimental evidence observing generation of photonic entanglement in enhanced green fluorescent protein (eGFP) [339] and exciton coupling between dimeric yellow fluorescent proteins, Venus, [340] strongly supports the hypothesis that FPs can interact coherently under physiological conditions. In this section, we review the theoretical background of excitation energy transfer and describe recent experimental progress in the study of quantum effects in FPs.…”

“…Moreover, physiological conditions are thought to promote rapid vibrational decoherence, thereby causing incoherent energy transfer [334] in which fluorophores behave independently, and excitation energy is transferred discretely from a donor to an acceptor. However, it has been observed that FPs show anomalous photophysical behaviour that cannot be explicitly explained by the theory of the very weak coupling [337][338][339][340][341][342][343][344][345][346], suggesting the possibility that excitation energy can be transferred coherently between FPs, even under physiological conditions.…”

A Guide to Fluorescence Lifetime Microscopy and Förster's Resonance Energy Transfer in Neuroscience

“…The rate of these energy hops is k FRET . Under specialized conditions, a donor and acceptor can be so strongly coupled to each other that they no longer behave independently (Förster, 1965; Kasha, 1963; Kenkre & Knox, 1974; kim et al, 2019). While still being composed of two fluorophores, quantum mechanically they behave as a single quantum entity.…”