An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching - PubMed (original) (raw)

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

Michele L Markwardt et al. PLoS One. 2011.

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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Conflict of interest statement

Competing Interests: The mutant CFPs described in this article are the topic of a pending patent application from the University of Maryland, Baltimore titled "Fluorescent Proteins and Uses Thereof" (SN 61/249,712). This patent covers the mutations used to derive mCerulean2 and mCerulean2.N variants that are the precursors to mCerulean3. Although the authors are pursuing commercial licensing and sale of their CFP reagents through companies like Clontech and Life Technologies, this does not alter their acceptance and adherence to the PLoS ONE policy as well as National Institutes of Health (NIH) policy for reagent sharing. All reagents described in the article are freely available upon reasonable request for the purpose of academic, non-commercial research, which will likely include deposition of the plasmids encoding mCerulean3 in a repository such as addgene.org.

Figures

Figure 1

Figure 1. Optimization of Cerulean.

A site-directed mutagenesis strategy was employed to optimize Cerulean fluorescence. (A) Residues on β-strand 7 (S147, D148; red), β-strand 8 (L166, I167, R168, H169; green) in the Cerulean X-ray structure (2wso.pdb [27]) were targeted for optimization by site-directed mutagenesis. The chromophore is colored blue. (B) T203 (orange) was targeted for optimization due to its proximity to the chromophore. T65 (green) was also mutated.

Figure 2

Figure 2. Spectral properties of new CFPs.

Absorption (dashed lines) and emission spectra (solid lines) are shown for Cerulean (black), mCerulean2 (green), mCerulean2.N (red), and mCerulean3 (blue). Spectra were normalized to the peak absorption or emission values.

Figure 3

Figure 3. 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.

Figure 4

Figure 4. Fluorescence imaging of mCerulean3 fusion vectors.

Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) mCerulean3-EB3-7 (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.

Figure 5

Figure 5. Fluorescence photoswitching behavior of CFPs in living cells.

COS7 cells expressing the indicated CFP were examined by widefield microscopy. Cells were bleached to 50% of their initial fluorescence by continuous, high intensity illumination of the full field of view. Recovery of cellular fluorescence was examined 15 min following the bleaching period. Data indicates the mean % recovery of bleached fluorescence after 15 min (n = 20, two-tailed t-test, difference from 0, *** indicates P<0.001, mCerulean3 recovery was not statistically significant (ns) under the same test,P = 0.09, n = 20).

Figure 6

Figure 6. 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.

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