We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.

The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission max ‫؍‬ 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.It is generally accepted that the enormous variety of colors and fluorescent hues displayed by living organisms are determined by chromoproteins and low molecular weight pigments. As a rule, chromoproteins typically require a prosthetic group: a small nonpeptide molecule or metal ion, which binds to the protein and is essential for the chromogenic properties of the protein (1-6).The only known exception to this rule is green fluorescent protein (GFP) 1 from Aequorea victoria (7). In contrast to other naturally occurring fluorescent proteins, the fluorescence of GFP is due entirely to an internal interaction between amino acids within the protein; no other cofactors or prosthetic groups are required. GFP owes its intrinsic fluorescence to a contiguous Ser-Tyr-Gly sequence centrally located within its primary structure. Upon folding, the protein modifies the fluorophoreforming sequence to produce an extended aromatic system (8 -10), which imparts the characteristic green fluorescence to the mature protein. Due to these distinctive properties, GFP has enjoyed extensive use as a biological marker in vivo (11, 12). Recently we described six novel GFP-like fluorescent proteins (FP) from nonbioluminescent Anthozoa species (13). It therefore became clear that GFP-like proteins are not necessarily components of bioluminescent systems but may simply determine fluorescent coloration of animals.In one particular case, we have shown that a GFP-like FP is responsible for the bright green fluorescence of the tentacle tips in the sea anemone Anemonia majano. However, in another sea anemone, Anemonia sulcata, we found that, although the tentacle tips do exhibit an intense purple color they are not significantly fluorescent (Fig. 1). The similarities of the color localization patterns and the close phylogenetic relationship of these two species led us to hypothesize that A. sulcata contains a purple nonfluorescent GFP homolog in its tentacles. In the p...