| RRC ID |
84967
|
| 著者 |
Laskaratou D, Fernández GS, Coucke Q, Fron E, Rocha S, Hofkens J, Hendrix J, Mizuno H.
|
| タイトル |
Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor.
|
| ジャーナル |
Nat Commun
|
| Abstract |
Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.
|
| 巻・号 |
12(1)
|
| ページ |
2541
|
| 公開日 |
2021-5-5
|
| DOI |
10.1038/s41467-021-22816-7
|
| PII |
10.1038/s41467-021-22816-7
|
| PMID |
33953187
|
| PMC |
PMC8099864
|
| MeSH |
Anisotropy
Bacterial Proteins / metabolism
Biosensing Techniques
Escherichia coli / genetics
Escherichia coli / metabolism
Fluorescence Resonance Energy Transfer / methods*
HeLa Cells
Humans
Optical Imaging / methods*
|
| IF |
12.121
|
| リソース情報 |
| 遺伝子材料 |
pcDNA3-GeuSapVC2.60 (RDB20914)
pcDNA3-GeuSapVC3.60 (RDB20915)
pRSET-GeudaSapphire (RDB20916)
pRSET-GeuSV11.5 (RDB20917) |
