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...
Novel dual emission, pH-sensitive variants of the green fluorescent protein (GFP) have been constructed and are suitable for ratiometric emission measurements in vivo. This new class of GFPs, termed deGPFs, results from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine. deGFPs display pK(a) values ranging from 6.8 to 8.0 and emission that switches from a green form (lambda(max) approximately 515 nm) to a blue form (lambda(max) approximately 460 nm) with acidifying pH. In this report we analyze in most detail the deGFP1 variant (S65T/H148G/T203C, pK(a) approximately 8.0) and the deGFP4 variant (S65T/C48S/H148C/T203C, pK(a) approximately 7.3). In the following paper [McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], data obtained by ultrafast fluorescence upconversion spectroscopy can be described by a kinetic model that includes an excited-state proton-transfer pathway at high pH but not at low pH. Crystal structure analyses of deGFP1 at high-pH and low-pH conformations were performed to elucidate the basis for the dual emission characteristics. At low pH the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited-state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore. Comparative analysis suggests that the basis for dual emission is elimination of the wild-type proton-transfer network by the S65T substitution, a general reduction in hydrogen-bonding opportunities, and a concomitant increase in the hydrophobic nature of the chromophore environment resulting from the cysteine substitutions. We evaluated the suitability of the deGFP4 variant for intracellular pH measurements in mammalian cells by transient expression in PS120 fibroblasts. The responses of deGFP4 and a commercially available pH-sensitive dye, SNARF-1, to changes in pH were compared in the same cells. Results show that the dynamic range of the emission ratio change is comparable between the two pH sensors over the range examined. Two-photon excitation was found to elicit a better deGFP4 fluorescent signal above cellular autofluorescence when compared to conventional confocal microscopy. Given their favorable optical characteristics, suitable pK(a)'s for the physiological pH range, and suitability for ratiometric measurements, dual emission GFPs should make excellent probes for studying pH in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.