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...

The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).