Topology-dependent FRET efficiency in living cells via N–C swapping of fluorescent protein fusions
Abstract
Förster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. FRET efficiencies were higher with EYFP than with EGFP as the donor. These efficiencies were comparable between the nucleus and cytoplasm. AlphaFold2-based structural modeling suggested similar proximity between donor and acceptor fluorophores despite structurally heterogeneous and loosely constrained geometries. In contrast, ensemble FRET simulations suggested that dynamic conformational sampling amplifies the apparent asymmetry between donor-acceptor and acceptor-donor configurations observed in live-cell FRET measurements. Collectively, these results demonstrate that topological arrangement, rather than static structural geometry alone, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.
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BibTeX
@article{tanida2026topologydependent,
title = {{Topology-dependent FRET efficiency in living cells via N–C swapping of fluorescent protein fusions}},
author = {Takashi Tanida and Md. Royhan Gofur and Takayuki Nakajima},
journal = {Scientific Reports},
year = {2026},
month = oct,
doi = {10.1038/s41598-026-71699-5},
url = {https://www.nature.com/articles/s41598-026-71699-5},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Topology-dependent FRET efficiency in living cells via N–C swapping of fluorescent protein fusions
AU - Takashi Tanida
AU - Md. Royhan Gofur
AU - Takayuki Nakajima
PY - 2026
DA - 2026/10/06
JO - Scientific Reports
DO - 10.1038/s41598-026-71699-5
UR - https://www.nature.com/articles/s41598-026-71699-5
AB - Förster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. FRET efficiencies were higher with EYFP than with EGFP as the donor. These efficiencies were comparable between the nucleus and cytoplasm. AlphaFold2-based structural modeling suggested similar proximity between donor and acceptor fluorophores despite structurally heterogeneous and loosely constrained geometries. In contrast, ensemble FRET simulations suggested that dynamic conformational sampling amplifies the apparent asymmetry between donor-acceptor and acceptor-donor configurations observed in live-cell FRET measurements. Collectively, these results demonstrate that topological arrangement, rather than static structural geometry alone, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.
SN - 2045-2322
ER -