title: Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.
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.
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
authors.forename: David
authors.initials: D
authors.lastname: von Stetten
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Noirclerc-Savoye
authors.forename: Mickaël
authors.initials: M
authors.lastname: Lelimousin
authors.forename: Linda
authors.initials: L
authors.lastname: Joosen
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hink
authors.forename: Laura
authors.initials: L
authors.lastname: van Weeren
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.forename: Antoine
authors.initials: A
authors.lastname: Royant
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins