An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching

Michele L. Markwardt, Gert Jan Kremers, Catherine A. Kraft, Krishanu Ray, Paula J C Cranfill, Korey A. Wilson, Richard Day, Rebekka M. Wachter, Michael W. Davidson, Mark A. Rizzo

Research output: Contribution to journalArticle

141 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article numbere17896
JournalPLoS One
Volume6
Issue number3
DOIs
StatePublished - 2011

Fingerprint

Energy Transfer
Luminance
Quantum yield
energy transfer
Fluorescence
fluorescence
Energy transfer
Proteins
Fusion reactions
Cells
Site-Directed Mutagenesis
Fluorescence Microscopy
Mutagenesis
Fluorophores
fluorescent dyes
Experiments
site-directed mutagenesis
Chromophores
X-Rays
Amino Acids

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Markwardt, M. L., Kremers, G. J., Kraft, C. A., Ray, K., Cranfill, P. J. C., Wilson, K. A., ... Rizzo, M. A. (2011). An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS One, 6(3), [e17896]. https://doi.org/10.1371/journal.pone.0017896

An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. / Markwardt, Michele L.; Kremers, Gert Jan; Kraft, Catherine A.; Ray, Krishanu; Cranfill, Paula J C; Wilson, Korey A.; Day, Richard; Wachter, Rebekka M.; Davidson, Michael W.; Rizzo, Mark A.

In: PLoS One, Vol. 6, No. 3, e17896, 2011.

Research output: Contribution to journalArticle

Markwardt, ML, Kremers, GJ, Kraft, CA, Ray, K, Cranfill, PJC, Wilson, KA, Day, R, Wachter, RM, Davidson, MW & Rizzo, MA 2011, 'An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching', PLoS One, vol. 6, no. 3, e17896. https://doi.org/10.1371/journal.pone.0017896
Markwardt, Michele L. ; Kremers, Gert Jan ; Kraft, Catherine A. ; Ray, Krishanu ; Cranfill, Paula J C ; Wilson, Korey A. ; Day, Richard ; Wachter, Rebekka M. ; Davidson, Michael W. ; Rizzo, Mark A. / An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. In: PLoS One. 2011 ; Vol. 6, No. 3.
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