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

(f-)PALM/STORM comparison of mMaple, mClavGR2 and mEos2.

<p>(<b>A–C</b>) Images of Δ<i>cheW E.coli</i> expressing CheW fusion proteins at L-arabinose concentrations optimal for swarming. Images contain (<b>A</b>) 1086 mMaple-CheW localizations (<b>B</b>) 694 mClavGR2-CheW localizations and (<b>C</b>) 229 mEos2-CheW localizations. (<b>D</b>) The mean number of photons emitted by each construct per photoconversion event (error is standard error, <i>N</i> = 3 independent measurements from distributions consisting of 4,000–32,000 localizations). Scale bars are 500 nm. (<b>E</b>) Distribution of the number of localizations observed for Δ<i>cheW E. coli</i> cells containing CheW fusions to mMaple, mClavGR2, and mEos2. Greater than 96% of cells expressing either mEos2- or mClavGR2-CheW fusions have less than 500 localizations (boxed region), whereas greater than 50% of cells expressing mMaple-CheW fusions have more than 500 localizations.</p

Properties of mMaple and related variants.

a<p>Extinction coefficent (mM⁻¹ cm⁻¹) at peak absorbance wavelength in PBS (pH 7.4). Value in parentheses was determined at pH 4 and pH 10, respectively.</p>b<p>Product of ε and Φ in mM⁻¹ cm⁻¹. For comparison, the brightness of EGFP and mCherry are 34 mM⁻¹ cm⁻¹ and 16 mM⁻¹ cm⁻¹, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051314#pone.0051314-Shaner2" target="_blank">[53]</a>.</p>c<p>Data from McKinney <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051314#pone.0051314-Mckinney1" target="_blank">[30]</a>.</p

(f-)PALM/STORM characterization of the number of observed localizations and proteins per cell.

<p>(<b>A–B</b>) (f-)PALM/STORM images of fixed <i>E. coli</i> expressing cytoplasmic (<b>A</b>) mMaple (<i>N</i> = 1696 localizations), or (<b>B</b>) mEos2 (<i>N</i> = 472 localizations). Localizations are represented as normalized 2D Gaussian peaks with widths given by their theoretical localization precisions (left panels) and plotted as small markers grouped into clusters with adjacent spacing of 30 nm or less (right panels). Individual protein localizations are shown in grey whereas closely spaced localizations (<30 nm) are grouped into clusters of the same color (right panels). The bright field and conventional fluorescence images are shown for comparison (left panels, left and right inset respectively). Scale bars are 500 nm. (<b>C</b>) Average number of localizations per cell for each cytoplasmically expressed pcFP. (<b>D</b>) The distribution of cluster sizes (<30 nm interlocalization spacing) for cytoplasmically expressed pcFPs. (<b>E</b>) Average number of cytoplasmically expressed proteins per cell. Rather than counting each localization as a single molecule, we count each cluster of localizations (localizations spaced <30 nm) as a single protein. The dotted lines in (A–B) denote the <i>E. coli</i> cell boundary. Scale bars are 500 nm and 50 nm (zooms). Zooms in (A–B) show possible reversible photoswitching events of single proteins. Error is the standard deviation (N = 20 cells (mMaple), N = 17 cells (mClavGR2), N = 16 cells (mEos2)). The large error bars are primarily due to variation in protein expression between cells.</p