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This study demonstrates that the orientation and topological arrangement of fluorescent proteins significantly affect FRET (Förster resonance energy transfer) efficiency in living cells, independent of the distance between fluorophores. Researchers found that acceptor-donor configurations (mCherry-EGFP or mCherry-EYFP) consistently produced higher FRET efficiencies than donor-acceptor configurations (EGFP-mCherry or EYFP-mCherry) across different cellular compartments. AlphaFold2 structural modeling revealed that while fluorophore proximity remained similar between configurations, the geometrical arrangement and orientation were more favorable in acceptor-donor setups.
Why it matters
These findings have direct implications for designing more efficient intramolecular FRET-based biosensors, which are widely used tools for monitoring molecular interactions and cellular processes in real-time. Understanding that protein topology, not just distance, drives FRET efficiency could enable researchers to optimize biosensor design by strategically positioning fluorescent proteins to maximize signal output.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
Forster 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 also 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. These efficiencies were comparable between the nucleus and cytoplasm. An orientation index analysis showed that the acceptor-donor configuration is more favorable than the donor-acceptor configuration regardless of donor variants and subcellular localization. FRET efficiencies were also higher with EYFP than with EGFP as the donor. AlphaFold2-based structural modeling suggested similar proximity with structurally heterogeneous and loosely constrained geometry of donor and acceptor fluorophores. Collectively, these results demonstrate that topological arrangement, rather than simple distance considerations, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.
Source: Topology-dependent FRET efficiency in living cells via N-C swapping of fluorescent protein fusions