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

Joachim Goedhart, David von Stetten, Marjolaine Noirclerc-Savoye, Mickaël Lelimousin, Linda Joosen, Mark A. Hink, Laura van Weeren, Theodorus W.J. Gadella () and Antoine Royant ()
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Joachim Goedhart: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam
David von Stetten: Structural Biology Group, European Synchrotron Radiation Facility
Marjolaine Noirclerc-Savoye: CNRS, Institut de Biologie Structurale Jean-Pierre Ebel
Mickaël Lelimousin: Structural Bioinformatics and Computational Biochemistry Unit, University of Oxford
Linda Joosen: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam
Mark A. Hink: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam
Laura van Weeren: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam
Theodorus W.J. Gadella: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam
Antoine Royant: Structural Biology Group, European Synchrotron Radiation Facility

Nature Communications, 2012, vol. 3, issue 1, 1-9

Abstract: 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.

Date: 2012
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DOI: 10.1038/ncomms1738

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