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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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Identification of mTurquoise2 and spectroscopic characterization.(a) Comparison of the Ile146 position in all known structures of CFP, highlighting its weak interaction with the chromophore (ECFP: light grey; Cerulean: dark grey; SCFP3A: light blue; mTurquoise: dark blue). The position of Phe146 in mTurquoise2 is shown as transparent green by anticipation. (b) Fluorescence lifetime imaging of bacterial colonies expressing 146X variants of mTurquoise with fluorescence intensities (upper left), and fluorescence modulation-based lifetimes (upper right). Sequencing revealed the identity of the residue at position 146 (lower left), with the corresponding residue in mTurquoise depicted in grey. Colonies with phenylalanine at 146 show an increased fluorescence lifetime, which can be discerned in the lifetime histogram as a separate peak around 4 ns, whereas the lifetime of mTurquoise is around 3.8 ns. The lifetime image is pseudo-coloured according to the scale shown in the histogram (values in ns). (c) Normalized absorption and fluorescence emission spectra of mTurquoise2. (d) pH dependence of mTurquoise2 fluorescence. Each point corresponds to the average of three measurements and is represented with the standard deviation. Data point interpolation (dark grey) results in a pKa of 3.1.
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f4: Identification of mTurquoise2 and spectroscopic characterization.(a) Comparison of the Ile146 position in all known structures of CFP, highlighting its weak interaction with the chromophore (ECFP: light grey; Cerulean: dark grey; SCFP3A: light blue; mTurquoise: dark blue). The position of Phe146 in mTurquoise2 is shown as transparent green by anticipation. (b) Fluorescence lifetime imaging of bacterial colonies expressing 146X variants of mTurquoise with fluorescence intensities (upper left), and fluorescence modulation-based lifetimes (upper right). Sequencing revealed the identity of the residue at position 146 (lower left), with the corresponding residue in mTurquoise depicted in grey. Colonies with phenylalanine at 146 show an increased fluorescence lifetime, which can be discerned in the lifetime histogram as a separate peak around 4 ns, whereas the lifetime of mTurquoise is around 3.8 ns. The lifetime image is pseudo-coloured according to the scale shown in the histogram (values in ns). (c) Normalized absorption and fluorescence emission spectra of mTurquoise2. (d) pH dependence of mTurquoise2 fluorescence. Each point corresponds to the average of three measurements and is represented with the standard deviation. Data point interpolation (dark grey) results in a pKa of 3.1.

Mentions: Our structural analysis of CFP variants highlighted the rigidity of most of the residues surrounding the chromophore, except for Ile146, which shows a high variability in position and conformation between all four variants (Fig. 4a). Thus, we performed site-directed saturation mutagenesis at position 146, transformed bacteria and analysed colonies, using a recently developed fluorescence lifetime-based screening method10. A significant number of colonies exhibited a drastic decrease in lifetime, which showed the importance of that specific residue. We picked colonies with a high lifetime (around 4 ns), moderate lifetime (below 3.5 ns) and low lifetime (below 3 ns) and verified their lifetime in a second round of fluorescence lifetime analysis (Fig. 4b). Close inspection of the lifetimes showed that the mutants with 146F have an increased lifetime relative to mTurquoise. We also found N, G and Q at position 146, yielding a lifetime between 3.0 and 3.2 ns, and R and S, yielding a lifetime of 2.8 ns. The 146N corresponds to the residue in wild-type green fluorescent protein (GFP) and shows that this substitution is detrimental for the CFP lifetime. We chose to characterize the properties of mTurquoise-146F, mTurquoise-146G and mTurquoise-146S in more detail. We also performed mutagenesis at position 220, because of the markedly different position of Leu220 in mTurquoise compared with SCFP3A. Also, position 165 was targeted as a negative control, since Phe165 is, like Ile146, a hydrophobic residue located on the same side of the indole ring, but to the other side of the symmetry axis. We only observed variants with similar or lower lifetime. The effect of mutating position 165 on lifetime was strong (similar to 146), whereas the effect of mutagenesis of residue 220 was relatively mild (Supplementary Fig. S3).


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)

Identification of mTurquoise2 and spectroscopic characterization.(a) Comparison of the Ile146 position in all known structures of CFP, highlighting its weak interaction with the chromophore (ECFP: light grey; Cerulean: dark grey; SCFP3A: light blue; mTurquoise: dark blue). The position of Phe146 in mTurquoise2 is shown as transparent green by anticipation. (b) Fluorescence lifetime imaging of bacterial colonies expressing 146X variants of mTurquoise with fluorescence intensities (upper left), and fluorescence modulation-based lifetimes (upper right). Sequencing revealed the identity of the residue at position 146 (lower left), with the corresponding residue in mTurquoise depicted in grey. Colonies with phenylalanine at 146 show an increased fluorescence lifetime, which can be discerned in the lifetime histogram as a separate peak around 4 ns, whereas the lifetime of mTurquoise is around 3.8 ns. The lifetime image is pseudo-coloured according to the scale shown in the histogram (values in ns). (c) Normalized absorption and fluorescence emission spectra of mTurquoise2. (d) pH dependence of mTurquoise2 fluorescence. Each point corresponds to the average of three measurements and is represented with the standard deviation. Data point interpolation (dark grey) results in a pKa of 3.1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Identification of mTurquoise2 and spectroscopic characterization.(a) Comparison of the Ile146 position in all known structures of CFP, highlighting its weak interaction with the chromophore (ECFP: light grey; Cerulean: dark grey; SCFP3A: light blue; mTurquoise: dark blue). The position of Phe146 in mTurquoise2 is shown as transparent green by anticipation. (b) Fluorescence lifetime imaging of bacterial colonies expressing 146X variants of mTurquoise with fluorescence intensities (upper left), and fluorescence modulation-based lifetimes (upper right). Sequencing revealed the identity of the residue at position 146 (lower left), with the corresponding residue in mTurquoise depicted in grey. Colonies with phenylalanine at 146 show an increased fluorescence lifetime, which can be discerned in the lifetime histogram as a separate peak around 4 ns, whereas the lifetime of mTurquoise is around 3.8 ns. The lifetime image is pseudo-coloured according to the scale shown in the histogram (values in ns). (c) Normalized absorption and fluorescence emission spectra of mTurquoise2. (d) pH dependence of mTurquoise2 fluorescence. Each point corresponds to the average of three measurements and is represented with the standard deviation. Data point interpolation (dark grey) results in a pKa of 3.1.
Mentions: Our structural analysis of CFP variants highlighted the rigidity of most of the residues surrounding the chromophore, except for Ile146, which shows a high variability in position and conformation between all four variants (Fig. 4a). Thus, we performed site-directed saturation mutagenesis at position 146, transformed bacteria and analysed colonies, using a recently developed fluorescence lifetime-based screening method10. A significant number of colonies exhibited a drastic decrease in lifetime, which showed the importance of that specific residue. We picked colonies with a high lifetime (around 4 ns), moderate lifetime (below 3.5 ns) and low lifetime (below 3 ns) and verified their lifetime in a second round of fluorescence lifetime analysis (Fig. 4b). Close inspection of the lifetimes showed that the mutants with 146F have an increased lifetime relative to mTurquoise. We also found N, G and Q at position 146, yielding a lifetime between 3.0 and 3.2 ns, and R and S, yielding a lifetime of 2.8 ns. The 146N corresponds to the residue in wild-type green fluorescent protein (GFP) and shows that this substitution is detrimental for the CFP lifetime. We chose to characterize the properties of mTurquoise-146F, mTurquoise-146G and mTurquoise-146S in more detail. We also performed mutagenesis at position 220, because of the markedly different position of Leu220 in mTurquoise compared with SCFP3A. Also, position 165 was targeted as a negative control, since Phe165 is, like Ile146, a hydrophobic residue located on the same side of the indole ring, but to the other side of the symmetry axis. We only observed variants with similar or lower lifetime. The effect of mutating position 165 on lifetime was strong (similar to 146), whereas the effect of mutagenesis of residue 220 was relatively mild (Supplementary Fig. S3).

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
Related in: MedlinePlus