The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, non-fluorescent pocilloporin to 2.2 Angstrom resolution and a genetically engineered fluorescent variant to 2.4 Angstrom resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gin-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment.

The 2.1 angstrom crystal structure of the far-red fluorescent protein HcRed: Inherent conformational flexibility of the chromophore

We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1 resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu64-Tyr65-Gly66, was observed to adopt both a cis coplanar and a tran. non-coplanar conformation. As a result of these two con formations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the b-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible. 2005 Elsevier Ltd. All rights reserved.

Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage

Extension of the conjugated pi-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore. (c) 2006 Elsevier Inc. All rights reserved.

Are corals colorful?

Using in situ spectrometry data and visual system modeling, we investigate whether the colors conferred to the reef-building corals by GFP-like proteins would look colorful not only to humans, but also to fish occupying different ecological niches on the reef. Some GFP-like proteins, most notably fluorescent greens and nonfluorescent chromoproteins, indeed generate intense color signals. An unexpected finding was that fluorescent proteins might also make corals appear less colorful to fish, counterbalancing the effect of absorption by the photosynthetic pigments of the endosymbiotic algae, which might be a form of protection against herbivores. We conclude that GFP-determined coloration of corals may be an important factor in visual ecology of the reef fishes.

The 2.1 angstrom 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 2.1 angstrom 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. (c) 2006 Elsevier Ltd . All rights reserved.

Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome

The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling. Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves. Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome. A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified. Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseuclogenes) that are not present in mouse. A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain. The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Response of holosymbiont pigments from the scleractinian coral Montipora monasteriata to short-term heat stress

Heating the scleractinian coral, Montipora monasteriata (Forskal 1775) to 32 degrees C under < 650 mu mol quanta m(-2) s(-1) led to bleaching in the form of a reduction in Peridinin, xanthophyll pool, chlorophyll c(2) and chlorophyll a, but areal dinoflagellates densities did not decline. Associated with this bleaching, chlorophyll (Chl) allomerization and dinoflagellate xanthophyll cycling increased. Chl allomerization is believed to result from the interaction of Chl with singlet oxygen (O-1(2)) or other reactive oxygen species. Thermally induced increases in Chl allomerization are consistent with other studies that have demonstrated that thermal stress generates reactive oxygen species in symbiotic dinoflagellates. Xanthophyll cycling requires the establishment of a pH gradient across the thylakoid membrane. Our results indicate that, during the early stages of thermal stress, thylakoid membranes are intact. Different morphs of M. monasteriata responded differently to the heat stress applied: heavily pigmented coral hosts taken from a high-light environment showed significant reductions in green fluorescent protein (GFP)-like homologues, whereas nonhost pigmented high-light morphs experienced a significant reduction in water-soluble protein content. Paradoxically, the more shade acclimated cave morph were, based on Chl fluorescence data, less thermally stressed than either of the high-light morphs. These results Support the importance of coral pigments for the regulation of the light environment within the host tissue.

Trans-cis Isomerism and acylimine formation in DsRed chromophore models: Intrinsic rotation barriers

The chromophore of the red fluorescent protein DsRed contains an acylimine substituent to a GFP-like chromophore structure. The acylimine is formed from the trans peptide linkage between residues F65 and Q66 in immature DsRed, but has a cis configuration in the mature protein. The relationship between acylimine formation and trans–cis isomerization is unresolved. We have calculated bond rotation profiles for models of mature and immature DsRed chromophores using B3LYP DFT. The isomerization barrier is substantially reduced in acylimine-substituted models, providing prima facie evidence that acylimine formation precedes trans–cis isomerization in DsRed chromophores.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice ( Oryza sativa), maize ( Zea mays), and wheat ( Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley ( Hordeum vulgare) have tissue-specific expression patterns.

Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome

Zinc-finger-containing proteins can be classified into evolutionary and functionally divergent protein families that share one or more domains in which a zinc ion is tetrahedrally coordinated by cysteines and histidines. The zinc finger domain defines one of the largest protein superfamilies in mammalian genomes; 46 different conserved zinc finger domains are listed in InterPro (http://www.ebi.ac.uk/InterPro). Zinc finger proteins can bind to DNA, RNA, other proteins, or lipids as a modular domain in combination with other conserved structures. Owing to this combinatorial diversity, different members of zinc finger superfamilies contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover. Accordingly, mutations of zinc finger genes lead to aberrations in a broad spectrum of biological processes such as development, differentiation, apoptosis, and immunological responses. This study provides the first comprehensive classification of zinc finger proteins in a mammalian transcriptome. Specific detailed analysis of the SP/Kruppel-like factors and the E3 ubiquitin-ligase RING-H2 families illustrates the importance of such an analysis for a more comprehensive functional classification of large protein families. We describe the characterization of a new family of C2H2 zinc-finger-containing proteins and a new conserved domain characteristic of this family, the identification and characterization of Sp8, a new member of the Sp family of transcriptional regulators, and the identification of five new RING-H2 proteins.

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles.