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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.

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Photostability of recombinant CFPs.Agarose beads labeled with CFPs as indicated were imaged at 60 s                            intervals under low power illumination (45 µW/cm2). At                            5 min, the beads were continuously illuminated for 60 s (red bar). (A)                            Representative images from the experimental data set are shown in                            pseudocolor to represent bead intensity. The scale bar indicates 10                            µm. (B) Bead fluorescence was normalized to prebleached intensity                            and plotted versus time. Bars indicate SD (n>15 for all samples). (C)                            The reversible (white) and irreversible (blue) bleached fractions were                            quantified over the 20 min recovery period.
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pone-0017896-g003: Photostability of recombinant CFPs.Agarose beads labeled with CFPs as indicated were imaged at 60 s intervals under low power illumination (45 µW/cm2). At 5 min, the beads were continuously illuminated for 60 s (red bar). (A) Representative images from the experimental data set are shown in pseudocolor to represent bead intensity. The scale bar indicates 10 µm. (B) Bead fluorescence was normalized to prebleached intensity and plotted versus time. Bars indicate SD (n>15 for all samples). (C) The reversible (white) and irreversible (blue) bleached fractions were quantified over the 20 min recovery period.

Mentions: By convention, we measured the fluorescence decay times for beads labeled with CFPs under continuous illumination. mCerulean2 and mTurquoise behaved similarly to mCerulean; however, mCerulean3 was resistant to fluorescence decay under these conditions. We observed an ∼18-fold longer decay half time for mCerulean3 than mTurquoise. Nonetheless, measurement of fluorescence decay times under continuous illumination has generally not provided reproducible results between laboratories [20], [21], [33], and therefore may not be the most useful predictor of performance. In addition, measurement of the rate of decay under continuous illumination does not take into account that some of the fluorescence loss may be reversible [25] and not indicative of a true photobleach. To distinguish between reversible photoswitching and irreversible bleaching, we imaged beads labeled with recombinant fluorescent proteins at 1 min intervals to establish baseline fluorescence using a low-power illumination intensity that we have successfully used for observation of living cells expressing CFPs. During the imaging protocol we illuminated continuously for a 1 min period to observe the extent of fluorescence decrease, and resumed imaging at 1 min intervals to quantify the extent of reversible photoswitching (Figure 3A, B). For Cerulean, and to a lesser extent, mTurquoise, the reduction of fluorescence induced by the 1 min illumination period was highly variable (for Cerulean, −17.6±8.6% reduction; all values are mean ± standard deviation (SD)) (Figure 3C). In addition, reversible fluorescence photoswitching was observable for both mTurquoise and Cerulean, and accounted for roughly half of the fluorescence decrease observed for mTurquoise beads over the 1 min continuous illumination period. In contrast, mCerulean3 was refractory to fluorescence reduction (1%±0.4, n = 15). Interestingly, we did not observe reversible photoswitching in beads labeled with mCerulean2.N, although the extent of irreversible photobleaching was extensive (Figure 3C). Taken together, we find that recombinant mCerulean3 is exceptionally photostable compared to other Aequorea-derived CFPs.


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)

Photostability of recombinant CFPs.Agarose beads labeled with CFPs as indicated were imaged at 60 s                            intervals under low power illumination (45 µW/cm2). At                            5 min, the beads were continuously illuminated for 60 s (red bar). (A)                            Representative images from the experimental data set are shown in                            pseudocolor to represent bead intensity. The scale bar indicates 10                            µm. (B) Bead fluorescence was normalized to prebleached intensity                            and plotted versus time. Bars indicate SD (n>15 for all samples). (C)                            The reversible (white) and irreversible (blue) bleached fractions were                            quantified over the 20 min recovery period.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017896-g003: Photostability of recombinant CFPs.Agarose beads labeled with CFPs as indicated were imaged at 60 s intervals under low power illumination (45 µW/cm2). At 5 min, the beads were continuously illuminated for 60 s (red bar). (A) Representative images from the experimental data set are shown in pseudocolor to represent bead intensity. The scale bar indicates 10 µm. (B) Bead fluorescence was normalized to prebleached intensity and plotted versus time. Bars indicate SD (n>15 for all samples). (C) The reversible (white) and irreversible (blue) bleached fractions were quantified over the 20 min recovery period.
Mentions: By convention, we measured the fluorescence decay times for beads labeled with CFPs under continuous illumination. mCerulean2 and mTurquoise behaved similarly to mCerulean; however, mCerulean3 was resistant to fluorescence decay under these conditions. We observed an ∼18-fold longer decay half time for mCerulean3 than mTurquoise. Nonetheless, measurement of fluorescence decay times under continuous illumination has generally not provided reproducible results between laboratories [20], [21], [33], and therefore may not be the most useful predictor of performance. In addition, measurement of the rate of decay under continuous illumination does not take into account that some of the fluorescence loss may be reversible [25] and not indicative of a true photobleach. To distinguish between reversible photoswitching and irreversible bleaching, we imaged beads labeled with recombinant fluorescent proteins at 1 min intervals to establish baseline fluorescence using a low-power illumination intensity that we have successfully used for observation of living cells expressing CFPs. During the imaging protocol we illuminated continuously for a 1 min period to observe the extent of fluorescence decrease, and resumed imaging at 1 min intervals to quantify the extent of reversible photoswitching (Figure 3A, B). For Cerulean, and to a lesser extent, mTurquoise, the reduction of fluorescence induced by the 1 min illumination period was highly variable (for Cerulean, −17.6±8.6% reduction; all values are mean ± standard deviation (SD)) (Figure 3C). In addition, reversible fluorescence photoswitching was observable for both mTurquoise and Cerulean, and accounted for roughly half of the fluorescence decrease observed for mTurquoise beads over the 1 min continuous illumination period. In contrast, mCerulean3 was refractory to fluorescence reduction (1%±0.4, n = 15). Interestingly, we did not observe reversible photoswitching in beads labeled with mCerulean2.N, although the extent of irreversible photobleaching was extensive (Figure 3C). Taken together, we find that recombinant mCerulean3 is exceptionally photostable compared to other Aequorea-derived CFPs.

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