Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution

Abstract

The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-Å resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2 1 and contain a tetramer with 222 noncrystallographic symmetry in the asymmetric unit. The refined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162. The protein, which forms an obligatory tetramer in solution and in the crystal, is a squat rectangular prism comprising four protomers whose fold is extremely similar to that of the Aequorea victoria green fluorescent protein despite low (Ϸ23%) amino acid sequence homology. The monomer consists of an 11-stranded ␤ barrel with a coaxial helix. The chromophores, formed from the primary sequence -Gln-Tyr-Gly-(residues 66 -68), are arranged in a Ϸ27 DsRed, a bright red fluorescent protein recently cloned from a corallimorpharian of the Discosoma genus, has considerable potential to complement existing uses of the extremely popular Aequorea victoria green fluorescent protein (avGFP) (for reviews, see refs. 1 and 2). Several fluorescent proteins (FPs) homologous to avGFP have been discovered in Anthozoa representatives. They function in part to contribute to the natural coloration of their hosts, and͞or possibly as one means of protection against UV radiation (3-5). Of particular interest are red-emitting ( max Ͼ 580 nm) FPs. In addition to their use in multicolor tagging experiments, these could, in principle, be very helpful by avoiding natural cellular autofluorescence and by extending the range of resonance energy transfer-based experiments (1). In this application, fluorescence resonance energy transfer between pairs of FPs may find use in the detection of protein-protein interactions or other proximity-related phenomena in vivo. One such red-emitting FP is commercially available from CLONTECH under the trade name of DsRed.DsRed, a 28-kDa polypeptide, has essentially the same chromophore as avGFP, autocatalytically formed from an internal Gln-Tyr-Gly (residues 66-68) tripeptide (amino acid sequence numbering of wild-type protein) (4). The overall amino acid sequence homology to avGFP is low, about 23%; however, several amino acids in the immediate vicinity of the chromophore are strictly conserved and are probably essential for chromophore formation (e.g., Glu-215 and Arg-95, corresponding to avGFP Glu-222 and Arg-96). The broad excitation and emission bands have maxima at 558 and 583 nm, respectively (with a minor peak at 494 nm and a significant tryptophan peak at 280 nm) for a monomer extinction coefficient and fluorescence quantum yield at 558 nm of approximately 75,000 mol Ϫ1 ͞cm Ϫ1and 0.7, respectively (6). DsRed is an excellent fluorescence resonance energy transfer counterpart to the yellow fluorescent variants of avGFP, which have emission maxima of about 525 nm (7,8), and its emission is distinct from that of avGFP for double-labeling experiments.DsRe...

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

“…The chromophore entries for the TNT geometry library was derived using the program AM4 as described. 7 Shelx-97 32 was used in the final stages of refinement. The linkages among backbone nitrogen and alpha carbon of Met66 and carbonyl carbon of Ile65, were unrestrained from the beginning of refinement with Shelx-97.…”

“…Cyan and green fluorescent proteins contain a chromophore chemically identical to that of GFP 5 while yellow, orange, and red emission results from additional modifications to the polypeptide backbone adjacent to a GFP-like chromophore. [6][7][8][9][10][11][12][13][14] In DsRed, the peptide bond immediately preceding the chromophore is oxidized to an acylimine; consequently the conjugation of the chromophore extends over the polypeptide backbone, increasing both the excitation and emission maxima relative to GFP. 6,7 zRFP574 14 and eqFP611 contain a DsRed-like chromophore although in the latter case, the chromophore adopts a trans conformation instead of the more typical cis conformation.…”

Evidence for the Isomerization and Decarboxylation in the Photoconversion of the Red Fluorescent Protein DsRed

“…Therefore, it is most likely that the hydrogen-bonding network around the chromophore in the SR state of DsRed is substantially different from that in the R state due to its proposed trans configuration and the decarboxylation of E215, since the carboxylate of E215 fills the adjacent space near to the phenolic group of the chromophore. 36 Therefore, the efficient nonradiative deactivation of SR might be attributed to the different hydrogen-bonding network as well as the free volume around the chromophore, which allow the formation of the twisted configuration. Free rotation of the chromophore as the origin of the dimness of SR, however, is excluded, since SR shows similar high anisotropy and long rotational correlation times compared to R in time-resolved anisotropy measurement (see Figure 5).…”

An improved mRFP1 adds red to bimolecular fluorescence complementation

“…Fluorescent proteins commonly form head-to-tail dimers, which, depending on the protein, can interact to form tetramers 11 . Assuming that hrGFP also forms head-to-tail dimers, the different RFP fragments should stick out toward the opposing ends of the complex.…”