Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria)

Abstract

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.

Figure 1

Pigments of A. sulcata var. rufescens. (A) Localization of GFPs in the upper side, orange fluorescent protein in the underside, and reddish protein in the tips of tentacles under daylight (scale bar: 1 cm) and UV (366 nm) (B). (C) Kryo-section (20 μm) of a tentacle fixed in seawater/4% paraformaldehyde irradiated with UV (365 nm) shows the ectodermal localization of the fluorescent proteins. The orange fluorescent protein located in the underside of the tentacle shows a yellow-shifted emission. This shift most likely is induced by the fixation. The red fluorescence of the entoderm is produced by chlorophyll of zooxanthellae (scale bar: 0.5 mm). (D) Agar plate with colonies of E. coli expressing asFP499 under UV light (366 nm). (E) E. coli expressing asCP562 under daylight. (F) Schematic model of asCP562 tertiary structure (residues 4–135) obtained from comparative protein modeling. Yellow: β-strands; red: helix; white/blue: loops. With a probability of 77%, the model describes a structure with a relative mean standard deviation lower than 5 Å compared with the corresponding control structure (–39). The residues Met-63–Tyr-64–Gly-65 in the middle of the distorted helix stand for the putative chromophore. Figure was produced with rasmol (40).

Figure 2

Excitation (ex)/emission (em) spectra of fluorescent proteins isolated from A. sulcata (A) and cloned in E. coli (B). Absorption spectra of the red protein isolated from the animal (C) and cloned in E. coli (D).

Figure 3

Molecular masses of the proteins. (A) The purified GFPs asFP499 and asFP522 isolated from A. sulcata migrate as one band in SDS gels (silver staining). Cloned asFP499 has the same molecular mass. (B) The nonfluorescent red protein asCP562 and the orange fluorescent protein asFP595 show identical molecular masses. (Part 1) Separated chromatographically on a Superdex 75 column, the proteins asFP595 and asCP562 isolated from the animal and cloned asCP562 exhibit identical elution profiles. The elution profiles of asFP595 is representative for these proteins. All proteins show apparent molecular masses of 66 kDa under both physiological conditions (0.05 M phosphate buffer, 100 mM NaCl, pH 7) and denaturating conditions (6 M guanidine hydrochloride) (part 2). (Part 3) The proteins of asFP499/522 show an apparent molecular mass of 66 kDa under physiological conditions, which indicates a natural occurrence of dimers or trimers. (Part 4) Under denaturing conditions three peaks can be observed. The first peak contains high molecular aggregations. The 66-kDa peak corresponds to oligomers of asFP499/522. The fraction with an apparent molecular mass of 23 kDa indicates asFP499/522 monomers.

Figure 4

(A) The proteins asFP595 and asCP562 isolated from A. sulcata and cloned asCP562 exhibit orange fluorescence when they are blotted on a nitrocellulose membrane. (B) A second fluorescent band at twice the molecular mass of asFP595 becomes visible if the protein is denatured without β-mercaptoethanol. This band is not detectable for asCP562 denatured without β-mercaptoethanol. The proteins were excited with broadband UV radiation. Nitrocellulose membranes were photographed by using a 610-nm long-pass filter. (C) All fluorescent proteins on the membrane have an emission maximum at 595 nm.

Figure 5

Multiple alignment of the proteins asFP499 and asCP562 with GFP of A. victoria and a majority sequence obtained from an alignment of six Anthozoa fluorescent proteins. Boxes mark the matching residues. The numbering of the alignment is based on GFP of A. victoria. The schematic diagram in the third line shows key structure elements of GFP of A. victoria as β-strands, cap regions, and the fluorophore. The secondary structure of the regions corresponding to the β-strands of GFP was predicted for the proteins by using phd prediction (–36). Predicted β-strands are shaded with light gray (reliability >62.1%) and dark gray (reliability >78.5%).