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Structural but not functional conservation of an immune molecule: a tachylectin-like gene in Hydractinia.

Tachylectin-related proteins are a recently characterized group of pattern recognition molecules, functioning in the innate immunity of various animals, from the ancient sponges to vertebrates. Tachylectins are characterized by six internal tandem repeats forming beta-propeller domains. We have identified and characterized a tachylectin-related gene in the colonial marine hydroid, Hydractinia echinata. The predicted gene product, termed CTRN, contained an N-terminal signal peptide and had a well-conserved tachylectin-like structure. RT-PCR analyses revealed only post-metamorphic expression while no mRNA was detected during embryonic development or in planula larvae. Exposure of colonies to LPS under conditions known to activate an immune response in Hydractinia did not result in upregulation of the gene. In situ hybridization analysis of metamorphosed animals detected CTRN transcripts only in a small subpopulation of neurons and their precursor cells, localized in a ring-like structure around the mouth of polyps. The same ring-like structure of CTRN expressing neurons was also observed in young polyp buds, predicting the position of the future mouth. This type of expression pattern can hardly be attributed to an immune-relevant gene. Thus, despite high structural similarity to tachylectins, this cnidarian member of this group seems to be an exception to all other tachylectins identified so far as it seems to have no function in cnidarian innate immunity.

Amino Acid Sequence↗

An evolutionary conserved role of Wnt signaling in stem cell fate decision.

Wnt/Frizzled/ss-catenin-based signaling systems play diverse roles in metazoan development, being involved not only in the establishment of body axes in embryogenesis but also in regulating stem cell fate in mammalian post-embryonic development. We have studied the role the canonical Wnt cascade plays in stem cell fate determination in Hydractinia, a member of the ancient metazoan phylum Cnidaria, by analyzing two key molecules in this pathway, frizzled and ss-catenin, and blocking GSK-3. Generally, frizzled was expressed in cells able to divide but absent in post-mitotic, terminally differentiated cells such as nerve cells and nematocytes. Transcripts of frizzled were identified in all embryonic stages beginning with maternal transcripts in the oocyte. Following gastrulation and in the planula larva, frizzled expression concentrated in the central endodermal mass from which the first interstitial stem cells and their derivatives arise. In post-metamorphic development, high levels of frizzled transcripts were detected in interstitial stem cells. Activating downstream events of the Wnt-cascade in the post-metamorphic life phase by blocking GSK-3 with paullones induced recruitment of nematocytes and nerve cells from the pool of interstitial stem cells. Terminal differentiation was preceded by an initial burst of proliferation of frizzled-positive i-cells. In activated i-cells, ss-catenin appeared in the cytoplasm, later in the nucleus. It was subsequently again observed in the cytoplasm and eventually faded out during terminal differentiation. Our results suggest an ancient role of Wnt signaling in stem cell fate determination.

Adenine↗

Intracellular Ca2+ increase induces post-fertilization events via MAP kinase dephosphorylation in eggs of the hydrozoan jellyfish Cladonema pacificum.

Naturally spawned eggs of the hydrozoan jellyfish Cladonema pacificum are arrested at G1-like pronuclear stage until fertilization. Fertilized eggs of Cladonema undergo a series of post-fertilization events, including loss of sperm-attracting ability, expression of adhesive materials on the egg surface, and initiation of cell cycle leading to DNA synthesis and cleavage. Here, we investigate whether these events are regulated by changes in intracellular Ca2+ concentration and mitogen-activated protein kinase (MAP kinase) activity in Cladonema eggs. We found that MAP kinase is maintained in the phosphorylated form in unfertilized eggs. Initiation of sperm-induced Ca2+ increase, which is the first sign of fertilization, was immediately followed by MAP kinase dephosphorylation within a few minutes of fertilization. The fertilized eggs typically stopped sperm attraction by an additional 5 min and became sticky around this time. They further underwent cytokinesis yielding 2-cell embryos at approximately 1 h post-fertilization, which was preceded by DNA synthesis evidenced by BrdU incorporation into the nuclei. Injection of inositol 1,4,5-trisphosphate (IP3) into unfertilized eggs, which produced a Ca2+ increase similar to that seen at fertilization, triggered MAP kinase dephosphorylation and the above post-fertilization events without insemination. Conversely, injection of BAPTA/Ca2+ into fertilized eggs at approximately 10 s after the initiation of Ca2+ increase immediately lowered the elevating Ca2+ level and inhibited the subsequent post-fertilization events. Treatment with U0126, an inhibitor of MAP kinase kinase (MEK), triggered the post-fertilization events in unfertilized eggs, where MAP kinase dephosphorylation but not Ca2+ increase was generated. Conversely, preinjection of the glutathione S-transferase (GST) fusion protein of MAP kinase kinase kinase (Mos), which maintained the phosphorylated state of MAP kinase, blocked the post-fertilization events in fertilized eggs without preventing a Ca2+ increase. These results strongly suggest that all of the three post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, lie downstream of MAP kinase dephosphorylation that is triggered by a Ca2+ increase.

Animals↗

Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae.

In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark-light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark-light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark-light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae.

Animals↗

The effect of larval age on developmental changes in the polyp prepattern of a hydrozoan planula.

The larvae of many marine organisms including hydrozoans are lecithotrophic and will not feed until after metamorphosis. In hydrozoans the aboral region of the planula becomes the holdfast and stolon, while the oral region becomes the stalk and hydranth that grows out of the holdfast following metamorphosis. If metamorphosis is delayed, the portion of the planula allocated to form holdfast and stolon shrinks and the region that forms the hydranth increases in size. Planulae also have the ability to regenerate their polyp prepattern. When the aboral region of the planula that does not normally form a hydranth is isolated and metamorphosis is delayed, it acquires the capacity to form a hydranth from the holdfast. A relatively high proportion of entodermal cells of young planulae engage in DNA synthesis (BrdU labeling index); as planulae age, the labeling index falls close to zero. When the polyp prepattern is modified during planula regeneration, entodermal cells are induced to engage in DNA synthesis. If DNA synthesis is inhibited in planulae, the polyp prepattern changes during regeneration and age-related developmental changes in planula are inhibited, suggesting that DNA synthesis is a necessary part of the pattern respecification process.

Animals↗

Changes in the patterning of a hydroid colony.

It is a widely held view that colonial hydroids (Cnidaria, Hydroidomedusae) are formed on the basis of a repetition of uniform elements. The dominant opinion is that the equal spatial organisation of the colony exists during all stages of its development except the primary polyp, which develops from the settled larva. However, the complex structure and large dimensions of shoots in certain thecate species (subcl. Leptomedusae) suggest that the organisation of the primary shoot differs strongly from that of established colonies. The present study based on a thorough collection and examination of the collected material allowed to describe the entire sequence of the colony ontogeny in Hydrallmania falcata (Sertulariidae). The established shoots of this species are characterised by relatively large size, spiral arrangement of pinnate branches over the shoot stem, and hydranths arranged in one row along the upper side of branches. We showed that the primary shoot developing from the larva has much smaller dimensions and an alternate arrangement of hydranths. During further colony development the shoot organisation undergoes a gradual transformation ending with the emergence of large shoots with 'characteristic' species-specific features. The discovered sequence of changes in shoot patterning shows certain correlations with alterations of the growing tip dimensions. The dimensions of the growing tip seem to determine the patterning in accordance with the particular spatial location of the tip. This finding implies the necessity of a detailed reinvestigation of the entire colony development in thecate hydroids, which would make a significant contribution to the understanding of the morphogenetic evolution and patterning mechanisms within this group of colonial organisms.

Animals↗

A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis.

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.

Amino Acid Sequence↗

Evolutionary aspects of developmentally regulated helix-loop-helix transcription factors in striated muscle of jellyfish.

The function of basic helix-loop-helix (bHLH) proteins in cell differentiation was shown to be conserved from Drosophila to vertebrates, exemplified by the function of MyoD in striated muscle differentiation. In phylogeny striated muscle tissue appears first in jellyfish and the question of its evolutionary position is controversially discussed. For this reason we have studied the developmental role of myogenic bHLH genes in medusa development. Based on their dimerization ability, four genes of the bHLH family of transcription factors were isolated from the hydrozoan jellyfish Podocoryne carnea. While the proteins Id and Ash group with cognate family members from bilaterians, Net-like and JellyD1 could not be unequivocally classified. Id is expressed during the medusa budding process and in the adult medusa, Ash and Net-like are expressed in all life cycle stages from egg to adult medusa and JellyD1 is expressed in the blastula and gastrula stages, the planula larva, and in late medusa bud stages. The dimerization specificity, the expression pattern, and the conservation of two residues specific for a MyoD bHLH domain suggest that JellyD1 is related to an ancestral MyoD gene. Id, Net-like, and JellyD1 are either expressed in the entocodon or its derived tissues, the striated and smooth muscle of the bell. These findings strengthen the hypothesis that the entocodon is a mesoderm-like structure and that the common ancestor of Cnidaria and Bilateria was more complex in cell-type architecture and body organization than commonly thought.

Amino Acid Sequence↗

Apparent membrane pore-formation by Portuguese Man-of-war (Physalia physalis) venom in intact cultured cells.

Intracellular, ratiometric microfluorimetry with fura-2 reveals that low doses of Portuguese Man-of-war (Physalia physalis) venom cause a linear increase in intracellular calcium accumulation by cultured L-929 cells. The influx of calcium is preceded by a lag period that is relatively independent of venom concentration, except at very low concentrations. Electron micrographs of negatively stained preparations of membranes from venom-treated L-929 and GH(4)C(1) cells exhibit 10-80 nm diameter lesions. The number and diameter of these lesions correlate with venom concentration. The venom forms lesions in GH(4)C(1) cells at much lower concentrations than in L-929 cells. Osmotic protectants such as sucrose and polyethylene glycol (PEG), reduce the extent of lactate dehydrogenase (LDH) release from venom-treated cells with the higher molecular weight PEG causing a greater inhibition of LDH release than sucrose. These results imply that Man-of-war venom produces pore-like structures in the membranes of target cells, which leads to colloid osmotic swelling with subsequent release of intracellular proteins and cell lysis.

Animals↗

The effect of Portuguese Man-of-war (Physalia physalis) venom on calcium, sodium and potassium fluxes of cultured embryonic chick heart cells.

Portuguese Man-of-war venom markedly increases calcium (45Ca2+) influx into primary, cultured, embryonic chick heart cells. This action is dose-dependent, but is unaffected by organic calcium blockers (diltiazem, verapamil, nifedipine, nimodipine and mibefradil). On the other hand, certain trivalent (La3+, Gd3+) and divalent (Zn2+, Ni2+, Cu2+, Mn2+) metals inhibit venom-induced calcium influx. Sodium (22Na+) influx into chick heart cells is also significantly increased by Man-of-war venom. Flecainide does not block venom-induced sodium influx. The efflux of the potassium analogue, 86Rb+, from heart cells is also significantly increased by the venom. The venom, however, has little or no effect on rubidium (86Rb+) or 2-deoxy-D-[2-3H] glucose influx.

Animals↗

Portuguese Man-of-war (Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes.

Portuguese Man-of-war (Physalia physalis) nematocyst venom dose-dependently stimulates calcium (45Ca(2+)) influx into L-929, GH(4)C(1), FRL, and embryonic chick heart cells. Venom-induced calcium influx is not blocked by ouabain, vanadate, nor organic calcium channel blockers, but is blocked by transition metal cations, such as lanthanum and zinc. Venom-induced calcium influx is accompanied in a dose-dependent manner by the release of intracellular lactate dehydrogenase, indicating a loss in plasma membrane integrity and cytolysis. Concentrations of zinc that block 45Ca(2+) influx also block lactate dehydrogenase release. Lanthanum, which also blocks 45Ca(2+) uptake, does not neutralize the cytolytic activity of the venom, but rather inhibits the venom's cytolytic action at the level of the target cell plasma membrane. Our findings indicate that Man-of-war venom causes an influx of calcium into several different cells types, not just those of the cardiovascular system, and this influx likely occurs by permeabilizing the plasma membranes of cells.

Animals↗

Biology and ecology of the hydrocoral millepora on coral reefs.

Millepores are colonial polypoidal hydrozoans secreting an internal calcareous skeleton of an encrusting or upright form, often of considerable size. Defensive polyps protruding from the skeleton are numerous and highly toxic and for this reason millepores are popularly known as "stinging corals" or "fire corals." In shallow tropical seas millepore colonies are conspicuous on coral reefs and may be locally abundant and important reef-framework builders. The history of systematic research on the Milleporidae and the sister family Stylasteridae is rich and full with the works of early naturalists beginning with Linnaeus. Seventeen living millepore species are recognised. Marked phenotypic variation in form and structure of colonies is characteristic of the genus Millepora. The first published descriptions of the anatomy and histology of millepores were by H. N. Moseley in one of the Challenger Expedition reports. These original, detailed accounts by Moseley remain valid and, except for recent descriptions of the ultrastructure of the skeleton and skeletogenic tissues, have not needed much modification. Millepores occur worldwide on coral reefs at depths of between 1 and 40 m and their distribution on reefs is generally zoned in response to physical factors. Colonies may be abundant locally on coral reefs but usually comprise <10% of the overall surface cover. Growth rates of colonies are similar to the measured rates of branching and platelike scleractinian corals. Millepores are voracious zooplankton feeders and they obtain part of their nutrition from autotrophic sources, photosynthetic production by symbiotic zooxanthellae. Reproduction in millepores is characterised by alternation of generations with a well-developed polypoid stage that buds off planktonic medusae. Sexual reproduction is seasonal for known species and the medusae have a brief planktonic life. Asexual production is achieved by sympodial growth, the production of new skeleton and soft tissue along a growing edge or branch tip, and by the reattachment, regeneration and repair of damaged or broken colony fragments. The physiological and ecological responses of species of millepores are similar to those of the species of scleractinian corals over a broad range of natural and anthropogenic disturbances. Severe damage to colonies may occur during major storms. Delicately branching species are more susceptible than massive and bladed species. The ability of broken fragments to regenerate can ameliorate the extent of damage. Widespread bleaching and mortality of millepores has been reported during mass bleaching events that have affected many coral reefs. Millepores are often the first to recover after short-term bleaching events. Harmful effects of oil spills, chronic oil pollution and oil-spill detergents have been widely reported for millepores. Although the hydrozoan coenosarc, with its fiercely stinging zooids, does not appear to be an attractive substratum for attachment and settlement of epizooans, a number of sessile and errant forms commonly occur on millepores. These include barnacles, amphipods, tanaid and alpheid crustaceans, polychaetes and gastropods. Burrowing molluscs, polychaetes and crustacea also abound. Many of these species or their close relatives also occur on scleractinian corals. A variety of predators, grazers and fouling organisms occur on millepores. These include errant polychaetes, several coral-feeding fish and a gastropod mollusc. Various invasive green, red and brown algae are widespread, growing on dead branches of millepores and overgrowing live coral tissue. Various "band diseases" associated with microorganisms that appear to cause lesions on millepores and loss of tissue have been documented but are not of widespread occurrence. Infestations of endolithic algae and fungi growing within the skeletons have been reported in a number of millepore species.

Animals↗

Fatal Portuguese man-o'-war (Physalia physalis) envenomation.

A fatal case of Physalia physalis (Portuguese man-o'-war) envenomation occurred on the Florida Atlantic coast in 1987. Despite appropriate beachside first aid, the patient was conscious only several minutes before having primary respiratory arrest and, later, cardiovascular collapse that resulted in death. Discharged nematocysts were still visible on the injured stratum corneum five days after envenomation. Additional treatment maneuvers suggested by this case include testing the tentacle fragments found on the victim's skin before their removal to ensure that nematocyst firing has been counteracted. We document the first human fatality caused by P physalis envenomation.

Aged↗

Violet bioluminescence and fast kinetics from W92F obelin: structure-based proposals for the bioluminescence triggering and the identification of the emitting species.

Obelin from the hydroid Obelia longissima and aequorin are members of a subfamily of Ca(2+)-regulated photoproteins that is a part of the larger EF-hand calcium binding protein family. On the addition of Ca(2+), obelin generates a blue bioluminescence emission (lambda(max) = 485 nm) as the result of the oxidative decarboxylation of the bound substrate, coelenterazine. The W92F obelin mutant is noteworthy because of the unusually high speed with which it responds to sudden changes of [Ca(2+)] and because it emits violet light rather than blue due to a prominent band with lambda(max) = 405 nm. Increase of pH in the range from 5.5 to 8.5 and using D(2)O both diminish the contribution of the 405 nm band, indicating that excited state proton transfer is involved. Fluorescence model studies have suggested the origin of the 485 nm emission as the excited state of an anion of coelenteramide, the bioluminescence reaction product, and 405 nm from the excited neutral state. Assuming that the dimensions of the substrate binding cavity do not change during the excited state formation, a His22 residue within hydrogen bonding distance to the 6-(p-hydroxy)-phenyl group of the excited coelenteramide is a likely candidate for accepting the phenol proton to produce an ion-pair excited state, in support of recent suggestions for the bioluminescence emitting state. The proton transfer could be impeded by removal of the Trp92 H-bond, resulting in strong enhancement of a 405 nm band giving the violet color of bioluminescence. Comparative analysis of 3D structures of the wild-type (WT) and W92F obelins reveals that there are structural displacements of certain key Ca(2+)-ligating residues in the loops of the two C-terminal EF hands as well as clear differences in hydrogen bond networks in W92F. For instance, the hydrogen bond between the side-chain oxygen atom of Asp169 and the main-chain nitrogen of Arg112 binds together the incoming alpha-helix of loop III and the exiting alpha-helix of loop IV in WT, providing probably concerted changes in these EF hands on calcium binding. But this linkage is not found in W92F obelin. These differences apparently do not change the overall affinity to calcium of W92F obelin but may account for the kinetic differences between the WT and mutant obelins. From analysis of the hydrogen bond network in the coelenterazine binding cavity, it is proposed that the trigger for bioluminescence reaction in these Ca(2+)-regulated photoproteins may be a shift of the hydrogen bond donor-acceptor separations around the coelenterazine-2-hydroperoxy substrate, initiated by small spatial adjustment of the exiting alpha-helix of loop IV.

Animals↗

Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation.

The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 Calpha planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 A resolution), the entirely green DsRed K70M mutant protein (1.9 A resolution), and the DsRed designed mutant Q66M (1.9 A resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp(2)-hybridized Gln 66 Calpha with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp(3)-hybridized Gln 66 Calpha with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization.

Acylation↗

Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry.

The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.

Alanine↗

Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination.

Global replacements of tyrosine by 2- and 3-fluorotyrosine in "enhanced green" and "enhanced yellow" mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with novel biophysical properties. While crystallographic and modeled structures of these proteins are indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP chromophore induces changes in the titration curves, variations in the intensity of the absorbance and fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context of the rigid chromophore-microenvironment interactions. The availability of the isomorpohous crystal structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl moiety exhibit a single conformation, while in the meta position two conformer states were observed in the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in "atomic mutations" (i.e., H --> F replacements) in the structures, offering unprecedented opportunities to understand and manipulate the relationships between protein structure and spectroscopic properties.

Animals↗