Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina

Abstract

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

Figure 1. A. equina specimen

The arrowheads point to the blue stripe on the basal disc that appears black in this black-and-white image.

Figure 2. Amino acid sequence alignment of the new chromoprotein aeCP597 and a number of GFP homologues

Sequence numbering corresponds to aeCP597 above the alignments and GFP beneath the alignments. Introduced gaps are represented by dashes. The residues with side chains forming the interior of the β-can are shaded in grey. Chromophore-forming residues are indicated by the dashed box. Mutated positions in aeCP597 sequence are boxed, and corresponding substitutions are shown above the boxes. In asFP595 and hcCP (Heteractis crispa non-fluorescent chromoprotein) sequences, amino acids that were mutated to produce red fluorescent mutants AsRed2 and HcRed1 (Clontech) are underlined. eqFP611, far-red fluorescent protein from the sea anemone Entacmaea quadricolor.

Figure 3. Spectral properties of aeCP597 and its fluorescent mutants

(A) Absorbance spectra for wild-type aeCP597 (solid line) and for the blue-coloured basal disc of the A. equina specimen (dotted line). (B) Excitation (dotted line) and emission (solid line) spectra for the AQ14 mutant. (C) Time-course of AQ14 absorbance spectrum change. Curve 1, freshly purified protein sample; curves 2–4, the same protein sample after 21, 62 and 183 h at 30 °C. (D) Excitation (dotted line) and emission (solid line) spectra for the AQ143 mutant.

Figure 4. Protein gel electrophoresis analysis of aeCP597 and its mutant AQ14 (Coomassie Blue staining)

Lanes 1 and 2, heated and unheated samples of recombinant purified aeCP597. Lines 3 and 4, heated and unheated samples of AQ14 after the ‘red-into-green’ spontaneous conversion. Positions of molecular-mass markers are shown on the left (sizes in kDa). The asterisk marks bands that are coloured/fluorescent before staining and correspond to native tetrameric protein in the unheated samples. The black triangle shows denatured full-length protein bands. The white triangles indicate positions of minor bands corresponding to 19 and 8 kDa fragments of broken polypeptide chain.

Figure 5. Confocal image of a HeLa cell expressing AQ143 (A) and emission spectrum of this cell measured using a 543-nm excitation laser line (B)

Scale bar in (A), 10 μm.