Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%
By J. Goedhart, D. von Stetten, M. Noirclerc-Savoye, M. Lelimousin, L. Joosen, M. Hink, Laura van Weeren, T. Gadella, A. Royant. Nature Communications, 2012
756 citations recorded by the source corpus.
Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein. Cyan variants of green fluorescent protein (CFPs) are widely used as donors in FRET experiments. Here, a new CFP, mTurquoise2, is developed, which displays a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime.
Subfields: Biophysics, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/bb0a2d1906545256568b19e6957ac28b59bee166
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Citations in the corpus
- Improving FRET dynamic range with bright green and red fluorescent proteins (2012, neutral): “16 ). mTurquoise1 and mTurquoise2 are recently described CFPs with dramatically increased quantum yields 30,43 , which would be expected to improve r 0 .”
- A Guide to Fluorescent Protein FRET Pairs (2016, background): “One advantage of cyan donors is that a few engineered CFPs have high quantum yield, including mTurquoise2 (QY = 0.93), mCerulean3 (QY = 0.87), mTFP1 (QY = 0.85) and Aquamarine (QY = 0.89) [34–37], which well-matches their large fluorescence lifetime (Table 2).”
- Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells (2017, methodology): “Previously, we have described lifeact fusions with mTurquoise2 (Goedhart et al., 2012) or mScarlet (Bindels et al., 2016).”