No abstract

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation--such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 degrees C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

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...

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.