A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria)

Abstract

We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4 -10. Red fluorophore maturation reaches a level of 90% after Ϸ12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS͞ PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture.G reen fluorescent protein from Aequorea victoria (avGFP) and its derivatives have become extremely popular in life science research as protein labels, markers of gene expression, and reporters of environmental conditions in living cells (1-3). Molecular properties of avGFP have been modified by sitedirected mutagenesis to optimize their applicability. Still, some properties, most importantly the restricted range of emission wavelengths, pose severe limitations in certain applications. In particular, red fluorescent proteins (FP) would be highly desirable because of the reduced cellular autofluorescence in the red spectral range and for use in multicolor labeling or fluorescence resonance energy transfer experiments. Efforts to produce stable red-fluorescent avGFP variants have not yet met with success. Recently, however, avGFP homologs were discovered in nonbioluminescent Anthozoa species (4-8). Some of the Anthozoa FPs show spectral properties that deviate greatly from avGFP, including red-emitting variants.One of them, a red FP from the corallimorpharian Discosoma sp. named drFP583 (6), commercially known as DsRed, became the subject of various biochemical and biophysical studies (9-12). These studies revealed detrimental properties for certain applications of the wild-type protein, especially the slow and incomplete maturation of the fluorophore, the obligate formation of tetramers, and the tendency to aggregate. Meanwhile, maturation properties have been greatly improved by engineering the protein, and the tendency to form aggregates was also reduced by mutagenesis (13-15). Recently, the oligomerization problem of DsRed has been solved by mutagenetic means (16).We have continued ...

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

“…At present, the most red shifted emission maximum of naturally occurring FPs is found in eqFP611 from the sea anemone Entacmaea quadricolor (16). A further red shift of fluorescence could be achieved in engineered variants of red fluorescent proteins or non-fluorescent chromoproteins (20).…”

“…Finally, oxidation of the amide nitrogen and Ca of the first amino acid of the chromogenic triad yields an acylimine bond that conjugates to the p-HBI system and causes the red-shifted fluorescence of proteins such as dsRed or eqFP611 (16,20,55,(52)(53)(54).…”

“…In some cases, the apparent aggregation was due to the accumulation of the marker protein in lysosomes (96). The tendency to form aggregates varies among different natural RFPs, ranging from pronounced in dsRed to virtually absent in eqFP611 (16,46). For several fusion proteins, the aggregation tendency of the marker prevented the correct localization of the fusion protein (97).…”

“…Naturally occurring red fluorescent GFP-like proteins were discovered in sea anemones (13). Shortly thereafter, the first genes of GFP-like proteins, including the red emitter dsRed, were isolated from different anthozoa species (14)(15)(16). Characterization of the novel proteins revealed that more than 700 million years of molecular evolution created diverse properties with exciting application potential, including an entire rainbow of fluorescence colors and the possibility to control the emission intensity or color by targeted light irradiation (8,(17)(18)(19)(20).…”

“…Characterization of the novel proteins revealed that more than 700 million years of molecular evolution created diverse properties with exciting application potential, including an entire rainbow of fluorescence colors and the possibility to control the emission intensity or color by targeted light irradiation (8,(17)(18)(19)(20). Unfortunately, adverse properties such as oligomerization or slow maturation may hamper the use of these proteins in some applications (16). In this review, we summarize biochemical and photophysical properties of GFP-like proteins and their relevance for imaging applications as well as prospects for their further optimization.…”