The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation

The crystal structure of a colorless variant of green fluorescent protein (GFP) containing the Y66L substitution has been determined to 1.5 A. Crystallographic evidence is presented for the formation of a trapped intermediate on the pathway of chromophore maturation, where the peptide backbone of residues 65-67 has condensed to form a five-membered heterocyclic ring. The hydroxyl leaving group remains attached to the ring as confirmed by high-resolution electrospray mass spectrometry. The alpha-carbon of residue 66 exhibits trigonal planar geometry, consistent with ring oxidation by molecular oxygen. Side chain positions of surrounding residues are not perturbed, in contrast to structural results obtained for the GFPsol-S65G/Y66G variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. The data are in accord with a reaction pathway in which dehydration is the last of three chemical steps in GFP chromophore formation. A novel mechanism for chromophore biosynthesis is proposed: when the protein folds, the backbone condenses to form a cyclopentyl tetrahedral intermediate. In the second step, the ring is oxidized by molecular oxygen. In the third and final step, elimination of the hydroxyl leaving group as water is coupled to a proton transfer reaction that may proceed via hydrogen-bonded solvent molecules. Replacement of the aromatic Tyr66 with an aliphatic residue appears to have a profound effect on the efficiency of ring dehydration. The proposed mechanism has important implications for understanding the factors that limit the maturation rate of GFP.     

Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments.

Many methods for detecting model genetically engineered microorganisms (GEMs) in experimental ecosystems rely on cultivation of introduced cells. In this study, survival of Escherichia coli was monitored with the green fluorescent protein (GFP) gene. This approach allowed enumeration of GEMs by both plating and microscopy. Use of the GFP-marked GEMs revealed that E. coli persisted in stream water at higher densities as determined microscopically than as determined by CFU enumeration. The GFP gene did not negatively impact the fitness of the host strain.     

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.     

Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein

In the interphase nuclei of cultured cells, chromatin is compacted and organized in higher-order structures through the condensation and decondensation processes. Chromosomes in the interphase nucleus are known to occupy distinct territories. The chromosome territory-interchromatin compartment model premises that the interchromatin compartment is separated from compact higher-order chromatin domains and expands in between these chromatin-organized territories. Chromatin in cultured cells is compacted under some conditions, such as the stress of heat shock and high osmolarity, and Src-mediated nuclear tyrosine phosphorylation. We report here that a novel arginine-rich cationic protein is generated by frameshift mutation of enhanced green fluorescent protein (EGFP). The arginine-rich cationic protein is highly hydrophilic and contains potential arginine-based nuclear localization signals. Expression of the arginine-rich cationic protein shows its predominant localization to the nucleus and induces striking chromatin condensation in the interphase, which might be involved in interchromatin spacing or euchromatinization. Thus, the arginine-rich cationic protein as a new tool would be useful for dissecting chromatin architecture dynamics.     

The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.     

Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution

We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.     

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.     

Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan-e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His(6) tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His(5) antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.     

Morphology and primary crystal structure of a silk-like protein polymer synthesized by genetically engineered Escherichia coli bacteria

The morphology and primary crystal structure of SLPF, a protein polymer produced by genetically engineered Escherichia coli bacteria, were characterized. SLPF is a segmented copolymer consisting of amino acid sequence blocks modeled on the crystalline segments of silk fibroin and the cell attachment domain of human fibronectin. Wide angle x-ray scattering (WAXS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and molecular simulations were used to analyze the primary crystal structure of SLPF. TEM experiments conducted on SLPF droplets cast from formic acid on amorphous carbon film demonstrated that these protein films have a microstructure formed of woven sheaves. The sheaves are composed of well-defined whisker crystallites. The width of the whiskers, 11.8 ± 2.2 nm, may be correlated to the length of the silk-like segment in SLPF as predicted by molecular simulations. WAXS data, TEM images, SAED, patterns, molecular simulations, and theoretical diffraction patterns all were consistent with the crankshaft model proposed for Silk I by Lotz and Keith. © 1994 John Wiley & Sons, Inc.     

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (λ_max = 510 nm) when excited with ultraviolet (UV) or blue light (λ_max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802–805; Stearns, Curr. Biol. 5 (1995) 262–264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501–12504] in a process involving cyclization of a Tyr⁶⁶ aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151–154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with redshifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (λ_max = 505 nm), but has a single excitation peak (λ_max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389–396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.     

Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil

The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.     

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

Superresolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Superresolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photoconvertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and reactivated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.     

The 2.1A crystal structure of copGFP, a representative member of the copepod clade within the green fluorescent protein superfamily

The green fluorescent protein (avGFP), its variants, and the closely related GFP-like proteins are characterized structurally by a cyclic tri-peptide chromophore located centrally within a conserved beta-can fold. Traditionally, these GFP family members have been isolated from the Cnidaria although recently, distantly related GFP-like proteins from the Bilateria, a sister group of the Cnidaria have been described, although no representative structure from this phylum has been reported to date. We have determined to 2.1A resolution the crystal structure of copGFP, a representative GFP-like protein from a copepod, a member of the Bilateria. The structure of copGFP revealed that, despite sharing only 19% sequence identity with GFP, the tri-peptide chromophore (Gly57-Tyr58-Gly59) of copGFP adopted a cis coplanar conformation within the conserved beta-can fold. However, the immediate environment surrounding the chromophore of copGFP was markedly atypical when compared to other members of the GFP-superfamily, with a large network of bulky residues observed to surround the chromophore. Arg87 and Glu222 (GFP numbering 96 and 222), the only two residues conserved between copGFP, GFP and GFP-like proteins are involved in autocatalytic genesis of the chromophore. Accordingly, the copGFP structure provides an alternative platform for the development of a new suite of fluorescent protein tools. Moreover, the structure suggests that the autocatalytic genesis of the chromophore is remarkably tolerant to a high degree of sequence and structural variation within the beta-can fold of the GFP superfamily.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95? by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal β-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded β-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.     

Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP.