Abstract
Yellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. Here, we characterize the yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with sub-diffraction resolution using STED nanoscopy and is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP is valuable FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live cell imaging including FRET experiments at acidic pH.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Funding This work was supported by LabEx PALM Grant ANR-10-LABX-0039-PALM and by the IDEX Paris Saclay (IRS BioProbe). H.V. was supported by a PhD felloship form MESRI.
The authors declare that they have no conflicts of interest with the contents of this article.
Supporting Information Available The file entitled Bousmah_ACSSensors_SI is available free of charge. It contains all details for plasmids construction and for in vitro expression and characterization of the recombinant proteins as well as supplementary figures and tables.
