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

Markwardt ML, Kremers GJ, Kraft CA, Ray K, Cranfill PJ, Wilson KA, Day RN, Wachter RM, Davidson MW, Rizzo MA - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

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.

Show MeSH

Related in: MedlinePlus

Improved FRET ratio imaging with mCerulean3.(A) To test the dependence of measured FRET ratios on illumination time,                            agarose beads were labeled with equivalent concentrations of the                            indicated CFP:mVenus fusion protein. Beads were imaged consecutively                            using constant illumination intensity (455 nm LED, 600                                µW/cm2), but a varied illumination period. Cyan and                            yellow fluorescence were captured simultaneously using an Optical                            Insights Dual-View containing standard CFP/YFP filter sets. FRET ratios                            were normalized to the peak FRET ratio. Points indicate the mean and                            error bars indicate SEM (n = 10). (B) HEK293 cells                            were transfected with the indicated fusion, and observed by fluorescence                            microscopy. The yellow/cyan FRET ratio of individual cells is shown                            (n = 50). Bar indicates the mean, and error bars                            indicate SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3066204&req=5

pone-0017896-g006: Improved FRET ratio imaging with mCerulean3.(A) To test the dependence of measured FRET ratios on illumination time, agarose beads were labeled with equivalent concentrations of the indicated CFP:mVenus fusion protein. Beads were imaged consecutively using constant illumination intensity (455 nm LED, 600 µW/cm2), but a varied illumination period. Cyan and yellow fluorescence were captured simultaneously using an Optical Insights Dual-View containing standard CFP/YFP filter sets. FRET ratios were normalized to the peak FRET ratio. Points indicate the mean and error bars indicate SEM (n = 10). (B) HEK293 cells were transfected with the indicated fusion, and observed by fluorescence microscopy. The yellow/cyan FRET ratio of individual cells is shown (n = 50). Bar indicates the mean, and error bars indicate SD.

Mentions: Many CFP:YFP FRET experiments utilize the ratio of the donor and acceptor fluorescence to detect changes induced by biosensing of cellular phenomena [36]; however, the instability and decay of CFP fluorescence over time could in theory affect the absolute FRET ratio if the decay in CFP fluorescence is shorter than the image capture time. To examine this in the context of FRET-ratio imaging, we labeled beads with recombinantly-generated CFP:YFP pairs, and examined the FRET ratio of beads over a broad range of image capture times using wide-field microscopy (Figure 6A). FRET ratios from fusions containing mCerulean or mTurquoise and mVenus were 10–20% less at long illumination times (∼1 s) compared with short ones (<1 ms) (Figure 6A). In contrast, FRET ratio measurements performed using mCerulean3 as a donor varied less than 2.5% as exposure time was varied over 4 orders of magnitude. To look at the impact of mCerulean3 photostability on FRET ratios observed in living cells, we expressed the same FRET fusion proteins containing mVenus that we used for the bead preparations in HEK cells (Figure 6B). Using mCerulean as the FRET donor, we observed FRET ratios ranging from 1 to 2.6 (Figure 6B), with a large SD of ±0.57 (n = 50). FRET ratios obtained for mTurquoise fusions were less variable (±0.37, n = 50), but the SD was still 2.5-fold greater than the results obtained with mCerulean3 fusions (±0.15, n = 50). Given that the improvement observed using mCerulean3 over mTurquoise is quite large compared to a small brightness advantage, it is likely that reduced photoswitching is the major component of the reduced variability. Thus, utilization of mCerulean3 as donor protein in FRET experiments reduces variability in ratio measurements.


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

Markwardt ML, Kremers GJ, Kraft CA, Ray K, Cranfill PJ, Wilson KA, Day RN, Wachter RM, Davidson MW, Rizzo MA - PLoS ONE (2011)

Improved FRET ratio imaging with mCerulean3.(A) To test the dependence of measured FRET ratios on illumination time,                            agarose beads were labeled with equivalent concentrations of the                            indicated CFP:mVenus fusion protein. Beads were imaged consecutively                            using constant illumination intensity (455 nm LED, 600                                µW/cm2), but a varied illumination period. Cyan and                            yellow fluorescence were captured simultaneously using an Optical                            Insights Dual-View containing standard CFP/YFP filter sets. FRET ratios                            were normalized to the peak FRET ratio. Points indicate the mean and                            error bars indicate SEM (n = 10). (B) HEK293 cells                            were transfected with the indicated fusion, and observed by fluorescence                            microscopy. The yellow/cyan FRET ratio of individual cells is shown                            (n = 50). Bar indicates the mean, and error bars                            indicate SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066204&req=5

pone-0017896-g006: Improved FRET ratio imaging with mCerulean3.(A) To test the dependence of measured FRET ratios on illumination time, agarose beads were labeled with equivalent concentrations of the indicated CFP:mVenus fusion protein. Beads were imaged consecutively using constant illumination intensity (455 nm LED, 600 µW/cm2), but a varied illumination period. Cyan and yellow fluorescence were captured simultaneously using an Optical Insights Dual-View containing standard CFP/YFP filter sets. FRET ratios were normalized to the peak FRET ratio. Points indicate the mean and error bars indicate SEM (n = 10). (B) HEK293 cells were transfected with the indicated fusion, and observed by fluorescence microscopy. The yellow/cyan FRET ratio of individual cells is shown (n = 50). Bar indicates the mean, and error bars indicate SD.
Mentions: Many CFP:YFP FRET experiments utilize the ratio of the donor and acceptor fluorescence to detect changes induced by biosensing of cellular phenomena [36]; however, the instability and decay of CFP fluorescence over time could in theory affect the absolute FRET ratio if the decay in CFP fluorescence is shorter than the image capture time. To examine this in the context of FRET-ratio imaging, we labeled beads with recombinantly-generated CFP:YFP pairs, and examined the FRET ratio of beads over a broad range of image capture times using wide-field microscopy (Figure 6A). FRET ratios from fusions containing mCerulean or mTurquoise and mVenus were 10–20% less at long illumination times (∼1 s) compared with short ones (<1 ms) (Figure 6A). In contrast, FRET ratio measurements performed using mCerulean3 as a donor varied less than 2.5% as exposure time was varied over 4 orders of magnitude. To look at the impact of mCerulean3 photostability on FRET ratios observed in living cells, we expressed the same FRET fusion proteins containing mVenus that we used for the bead preparations in HEK cells (Figure 6B). Using mCerulean as the FRET donor, we observed FRET ratios ranging from 1 to 2.6 (Figure 6B), with a large SD of ±0.57 (n = 50). FRET ratios obtained for mTurquoise fusions were less variable (±0.37, n = 50), but the SD was still 2.5-fold greater than the results obtained with mCerulean3 fusions (±0.15, n = 50). Given that the improvement observed using mCerulean3 over mTurquoise is quite large compared to a small brightness advantage, it is likely that reduced photoswitching is the major component of the reduced variability. Thus, utilization of mCerulean3 as donor protein in FRET experiments reduces variability in ratio measurements.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus