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Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.

Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW, Royant A - Nat Commun (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.

ABSTRACT
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.

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ECFP residues examined in this work.The secondary structure of ECFP is drawn in light grey, and the chromophore in cyan. Residues that have been mutated in the previously described improved variants are shown in yellow, and those mutated in the present work in green.
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f1: ECFP residues examined in this work.The secondary structure of ECFP is drawn in light grey, and the chromophore in cyan. Residues that have been mutated in the previously described improved variants are shown in yellow, and those mutated in the present work in green.

Mentions: Structural analysis of ECFP revealed that the mutations resulted in alternate conformations of the seventh strand of the β-barrel67. Rational design based on these alternate conformations led to an improved variant called Cerulean (QY=0.49), containing the two mutations Y145A and H148D8. Nevertheless, Cerulean still exhibited structural heterogeneity of the seventh strand7. A different study, employing site-directed mutagenesis to evaluate the effect of individual mutations, led to the development of Super Cyan Fluorescent Protein 3A (SCFP3A) (QY=0.56), which harbours only the H148D mutation9. Apart from moderate brightness, all these mutants suffer from complex fluorescence decay kinetics, complicating the analysis of lifetime-based FRET measurements. Recently, a family of much improved variants were identified by a combination of site-directed mutagenesis and a new fluorescence lifetime-based screening10. Surprisingly, it took only a single mutation of a residue that is part of the chromophore, T65S, to significantly improve the brightness of SCFP3A. The resulting variant, mTurquoise, is 50% brighter owing to its high QY of 0.84 and exhibits a mono-exponential fluorescence decay11. Another mutant, mTurquoise-148G/224L (mTurquoise-GL), exhibits one of the longest fluorescence lifetimes observed for any fluorescent protein. The residues of ECFP, which have been mutated to successively yield Cerulean, SCFP3A, mTurquoise and mTurquoise-GL, are highlighted in yellow in Fig. 1 and listed in Supplementary Table S1. Recently, improved CFP variants based on Cerulean were described12. mCerulean2 was obtained from Cerulean using several mutations, which include the critical H148G mutation, previously identified in mTurquoise-GL. Subsequently, mCerulean3 was considerably improved by the single mutation T65S, which is also responsible for the high QY of mTurquoise. The QYs were significantly improved to 0.60 and 0.87 for mCerulean2 and mCerulean3, respectively.


Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.

Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW, Royant A - Nat Commun (2012)

ECFP residues examined in this work.The secondary structure of ECFP is drawn in light grey, and the chromophore in cyan. Residues that have been mutated in the previously described improved variants are shown in yellow, and those mutated in the present work in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316892&req=5

f1: ECFP residues examined in this work.The secondary structure of ECFP is drawn in light grey, and the chromophore in cyan. Residues that have been mutated in the previously described improved variants are shown in yellow, and those mutated in the present work in green.
Mentions: Structural analysis of ECFP revealed that the mutations resulted in alternate conformations of the seventh strand of the β-barrel67. Rational design based on these alternate conformations led to an improved variant called Cerulean (QY=0.49), containing the two mutations Y145A and H148D8. Nevertheless, Cerulean still exhibited structural heterogeneity of the seventh strand7. A different study, employing site-directed mutagenesis to evaluate the effect of individual mutations, led to the development of Super Cyan Fluorescent Protein 3A (SCFP3A) (QY=0.56), which harbours only the H148D mutation9. Apart from moderate brightness, all these mutants suffer from complex fluorescence decay kinetics, complicating the analysis of lifetime-based FRET measurements. Recently, a family of much improved variants were identified by a combination of site-directed mutagenesis and a new fluorescence lifetime-based screening10. Surprisingly, it took only a single mutation of a residue that is part of the chromophore, T65S, to significantly improve the brightness of SCFP3A. The resulting variant, mTurquoise, is 50% brighter owing to its high QY of 0.84 and exhibits a mono-exponential fluorescence decay11. Another mutant, mTurquoise-148G/224L (mTurquoise-GL), exhibits one of the longest fluorescence lifetimes observed for any fluorescent protein. The residues of ECFP, which have been mutated to successively yield Cerulean, SCFP3A, mTurquoise and mTurquoise-GL, are highlighted in yellow in Fig. 1 and listed in Supplementary Table S1. Recently, improved CFP variants based on Cerulean were described12. mCerulean2 was obtained from Cerulean using several mutations, which include the critical H148G mutation, previously identified in mTurquoise-GL. Subsequently, mCerulean3 was considerably improved by the single mutation T65S, which is also responsible for the high QY of mTurquoise. The QYs were significantly improved to 0.60 and 0.87 for mCerulean2 and mCerulean3, respectively.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.

ABSTRACT
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.

Show MeSH