Conversion of red fluorescent protein into a bright blue probe

Abstract

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Figure 1

(A) Immediate environment of the chromophore in eqFP611 [12]. The chromophore is showed in black, the conserved amino acid residues are in light gray, and non-conserved residues are in gray. The hydrogen bonds are indicated with dashed lines. (B) Amino acid sequence alignment of mTagBFP with EGFP, Azurite, EBFP2, eqFP611 and TagRFP. Structurally important regions are highlighted in grey, beta-strands are shown with arrows, alpha-helixes are shown with ribbons. The chromophore forming residues are marked with asterisks. Site-specific mutations resulted in conversion of TagRFP into a blue fluorescent predecessor of mTagBFP are shown white on black background. Mutations generated in the course of random mutagenesis are shown white italic on dark grey background. The alignment numbering follows that for EGFP.

Figure 2

Spectral, biochemical and photobleaching properties of the purified mTagBFP. (A) Absorbance, excitation and emission spectra of mTagBFP. (B) Maturation kinetics for mTagBFP and EBFP2. (C) pH dependence for mTagBFP and EBFP2. (D) Photobleaching curves for purified mTagBFP (solid line), EBFP2 (short dashed line) and Azurite (dashed line) under mercury arc lamp illumination. (E) Photobleaching curves for mTagBFP (solid line), EBFP2 (short dashed line) or Azurite (dashed line) expressed in HeLa cells using 405 nm laser scanning. Data represents an average of 8–10 cells per each protein. The time axes in D and E represent the normalized imaging time with an initial emission rate of 1,000 photons/second per molecule.

Figure 3

Fluorescence lifetimes for mTagBFP, EBFP2 and Azurite. (A) Fluorescence decay for mTagBFP (dark grey squares), EBFP2 (light grey triangles) and Azurite (black circles) at pH 7.4 and 23°C. Solid lines indicate fits by the mono-exponential curves; (B) Dependence of fluorescence lifetimes for mTagBFP (dark grey), EBFP2 (light grey) and Azurite (black) on pH and temperature. pH 7.4, 23°C (filled bars); pH 6.0, 23°C (diagonal bars); pH 7.4, 37°C (diamond bars). Error does not exceed 5%.

Figure 4

Behavior of mTagBFP as the FRET donor. (A) FRET of mTagBFP-mTagGFP fusion construct in vitro. (B,C) FRET imaging of staurosporine-treated HeLa cells: (B) Time course of corrected FRET normalized per intensity of the donor observed in four cells indicated in panels (C). The values are normalized to time of the staurosporine addition. (C) Fluorescent images of the cells after staurosporine treatment are shown as overlaid images of blue and green channels in upper panels. The corrected FRET signals are shown as pseudocolor images in lower panels. Scale bar is 10 μm.