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.

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...

Recently, we cloned several fluorescent proteins of different colors homologous to Aequorea victoria green fluorescent protein, which have great biotechnological potential as in vivo markers of gene expression. However, later investigations revealed severe drawbacks in the use of novel fluorescent proteins (FPs), in particular, the formation of tetramers (tetramerization) and high molecular weight aggregates (aggregation). In this report, we employ a mutagenic approach to resolve the problem of aggregation. The elimination of basic residues located near the N-termini of FPs results in the generation of non-aggregating versions of several FPs, specifically, drFP583 (DsRed), DsRedTimer, ds/drFP616, zFP506, zFP538, amFP486, and asFP595. ß

Geometry configurations of a large fraction of the kindling fluorescent protein asFP595 around the chromophore region were optimized by using the effective fragment potential quantum mechanical-molecular mechanical (QM/MM) method. The initial coordinates of heavy atoms were taken from the structure from the Protein Data Bank archive corresponding to the dark-adapted state of the Ala143 --> Gly mutant of asFP595. Optimization of geometry parameters was performed for all internal coordinates in the QM part composed of the chromophore unit and the side chains of His197, Glu215, and Arg92 as well as for positions of effective fragments constituting the MMpart. The structures corresponding to the anion trans, anion cis, and zwitterion trans moieties were considered among various alternatives for the chromophore unit inside the protein matrix. The QM/MM simulations show that the protein environment provides stabilization for the trans-zwitterion isomer compared to the gas-phase conditions. By using the multiconfigurational CASSCF and the time-dependent density functional theory calculations, we estimated positions of spectral bands corresponding to vertical S(0)-S(1) transitions. The results of simulations support the assumption that the dark state of asFP595 corresponds to the anionic or zwitterionic trans-conformation, while the kindled state corresponds to the anionic cis-conformation.