An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

[1]  R. Wachter,et al.  Mechanistic diversity of red fluorescence acquisition by GFP-like proteins. , 2010, Biochemistry.

[2]  F. Mérola,et al.  Relation between pH, structure, and absorption spectrum of Cerulean: A study by molecular dynamics and TD DFT calculations , 2010, Proteins.

[3]  J. Goedhart,et al.  Bright cyan fluorescent protein variants identified by fluorescence lifetime screening , 2010, Nature Methods.

[4]  M. Davidson,et al.  Fluorescent protein tracking and detection: applications using fluorescent proteins in living cells. , 2009, Cold Spring Harbor protocols.

[5]  Antoine Royant,et al.  Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield. , 2009, Biochemistry.

[6]  E. Gratton,et al.  Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms , 2008, The Journal of cell biology.

[7]  P. Dorrestein,et al.  Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants. , 2008, Biochemistry.

[8]  Michael Z. Lin,et al.  Improving the photostability of bright monomeric orange and red fluorescent proteins , 2008, Nature Methods.

[9]  Ammasi Periasamy,et al.  Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. , 2008, Journal of biomedical optics.

[10]  E. Gratton,et al.  The phasor approach to fluorescence lifetime imaging analysis. , 2008, Biophysical journal.

[11]  M. Davidson,et al.  Advances in fluorescent protein technology , 2007, Journal of Cell Science.

[12]  Enrico Gratton,et al.  Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies , 2007, The Journal of cell biology.

[13]  Yuichiro Hori,et al.  [Crystal structure of the Aequorea victoria green fluorescent protein]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[14]  D. Piston,et al.  X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging. , 2007, Biochemistry.

[15]  Robert E Campbell,et al.  Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. , 2007, Biochemistry.

[16]  Joachim Goedhart,et al.  Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. , 2007, Biochemistry.

[17]  R. Wachter,et al.  Chromogenic cross-link formation in green fluorescent protein. , 2007, Accounts of chemical research.

[18]  Steven S. Vogel,et al.  Cerulean, Venus, and VenusY67C FRET reference standards. , 2006, Biophysical journal.

[19]  Jun O. Liu,et al.  Reading dynamic kinase activity in living cells for high-throughput screening. , 2006, ACS chemical biology.

[20]  W. Zipfel,et al.  Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins , 2006, Microscopy and Microanalysis.

[21]  Joachim Goedhart,et al.  UvA-DARE ( Digital Academic Repository ) Optimization of fluorescent proteins for novel quantitative multiparameter microscopy approaches , 2007 .

[22]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[23]  Jacqueline Ridard,et al.  Cyan fluorescent protein: molecular dynamics, simulations, and electronic absorption spectrum. , 2005, The journal of physical chemistry. B.

[24]  L. Langeberg,et al.  The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways , 2005, Nature.

[25]  R. Clegg,et al.  Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes , 2005, Journal of Fluorescence.

[26]  T. Zal,et al.  Using live FRET imaging to reveal early protein-protein interactions during T cell activation. , 2004, Current opinion in immunology.

[27]  T. Zal,et al.  Photobleaching-corrected FRET efficiency imaging of live cells. , 2004, Biophysical journal.

[28]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.

[29]  Kees Jalink,et al.  Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission. , 2004, Biophysical journal.

[30]  Marc Tramier,et al.  Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. , 2002, Biophysical journal.

[31]  M E Dickinson,et al.  Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. , 2001, BioTechniques.

[32]  R. Tsien,et al.  Reducing the Environmental Sensitivity of Yellow Fluorescent Protein , 2001, The Journal of Biological Chemistry.

[33]  M. Ohkura,et al.  A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein , 2001, Nature Biotechnology.

[34]  R. Tsien,et al.  Circular permutation and receptor insertion within green fluorescent proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[36]  R Y Tsien,et al.  Understanding, improving and using green fluorescent proteins. , 1995, Trends in biochemical sciences.

[37]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[38]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[39]  M. Karin,et al.  GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. , 1993, Genes & development.

[40]  W. M. Westler,et al.  Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. , 1993, Biochemistry.

[41]  A. Gloyn,et al.  Naturally occurring glucokinase mutations are associated with defects in posttranslational S-nitrosylation. , 2010, Molecular endocrinology.

[42]  D. Piston,et al.  FRET by fluorescence polarization microscopy. , 2008, Methods in cell biology.

[43]  K. Hahn,et al.  Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. , 2006, Methods in enzymology.

[44]  K. Mikoshiba,et al.  A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications , 2002, Nature Biotechnology.

[45]  R. Heim,et al.  Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. , 1999, Methods in cell biology.

[46]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[47]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[48]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .