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Protein Domains (protein + domain)
Selected AbstractsThe Photoreaction of the Photoactive Yellow Protein Domain in the Light Sensor Histidine Kinase Ppr is Influenced by the C-terminal Domains,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Hironari Kamikubo To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p- coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p -coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYPL/dark and PYPM/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYPL - and PYPM -like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYPL compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains. [source] Myosin diversity in the diatom Phaeodactylum tricornutum,CYTOSKELETON, Issue 3 2010Matthew B. Heintzelman Abstract This report describes the domain architecture of ten myosins cloned from the pennate diatom Phaeodactylum tricornutum. Several of the P. tricornutum myosins show similarity to myosins from the centric diatom Thalassiosira pseudonana as well as to one myosin from the oomycete Phytophthora ramorum. The P. tricornutum myosins, ranging in size from 126 kDa to over 250 kDa, all possess the canonical head, neck and tail domains common to most myosins, though variations in each of these domains is evident. Among the features distinguishing several of the diatom myosin head domains are N-terminal SH3-like domains, variations in or near the P-loop and Loop 1 regions close to the nucleotide binding pocket, and extended converter domains. Variations in the length of the neck domain or lever arm, defined by the light chain-binding IQ motifs, are apparent with the different diatom myosins predicted to contain from one to nine IQ motifs. Protein domains found within the P. tricornutum myosin tails include regions of coiled-coil structure, ankyrin repeats, CBS domain pairs, a PB1 domain, a kinase domain and a FYVE-finger motif. As many of these features have never before been characterized in myosins of any type, it is likely that these new diatom myosins will expand the repertoire of known myosin behaviors. © 2010 Wiley-Liss, Inc. [source] Dual fluorescent protein reporters for studying cell behaviors in vivoGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 10 2009M. David Stewart Abstract Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color-coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color-code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color-code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time-lapse movies of fluorescently-labeled primordial germs cells demonstrate the utility of the color-code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo. genesis 47:708,717, 2009. © 2009 Wiley-Liss, Inc. [source] Phylogenetic and transcriptional analysis of a strictosidine synthase-like gene family in Arabidopsis thaliana reveals involvement in plant defence responsesPLANT BIOLOGY, Issue 1 2009M. M. Sohani Abstract Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been uncovered in the genomes of all multicellular organisms sequenced so far and are known to play a role in animal immune responses. Among several distinct groups of Arabidopsis thaliana SSL sequences, four genes (AtSSL4,AtSSL7) arranged in tandem on chromosome 3 show more similarity to SSL genes from Drosophila melanogaster and Caenorhabditis elegans than to other Arabidopsis SSL genes. To examine whether any of the four AtSSL genes are immune-inducible, we analysed the expression of each of the four AtSSL genes after exposure to microbial pathogens, wounding and plant defence elicitors using real-time quantitative RT-PCR, Northern blot hybridisation and Western blot analysis with antibodies raised against recombinant AtSSL proteins. While the AtSSL4 gene was constitutively expressed and not significantly induced by any treatment, the other three AtSSL genes were induced to various degrees by plant defence signalling compounds, such as salicylic acid, methyl jasmonate and ethylene, as well as by wounding and exposure to the plant pathogens Alternaria brassicicola and cucumber mosaic virus. Our data demonstrate that the four SSL-coding genes are regulated individually, suggesting specific roles in basal (SSL4) and inducible (SSL5-7) plant defence mechanisms. [source] Oropharyngeal Skeletal Disease Accompanying High Bone Mass and Novel LRP5 Mutation,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2005Michael R Rickels Abstract Gain-of-function mutation in the gene encoding LRP5 causes high bone mass. A 59-year-old woman carrying a novel LRP5 missense mutation, Arg154Met, manifested skeletal disease affecting her oropharynx as well as dense bones, showing that exuberant LRP5 effects are not always benign. Introduction: Gain-of-function mutation (Gly171Val) of LDL receptor-related protein 5 (LRP5) was discovered in 2002 in two American kindreds with high bone mass and benign phenotypes. In 2003, however, skeletal disease was reported for individuals from the Americas and Europe carrying any of six novel LRP5 missense mutations affecting the same LRP5 protein domain. Furthermore, in 2004, we described a patient with neurologic complications from dense bones and extensive oropharyngeal exostoses caused by the Gly171Val defect. Materials and Methods: A 59-year-old woman was referred for dense bones. Three years before, mandibular buccal and lingual exostoses (osseous "tori") were removed because of infections from food trapping between the teeth and exostoses. Maxillary buccal and palatal exostoses were asymptomatic. Radiographic skeletal survey showed marked thickening of the skull base and diaphyses of long bones (endosteal hyperostosis). BMD Z scores assessed by DXA were +8.5 and +8.7 in the total hip and L1 -L4 spine (both ,195% average control), respectively. LRP5 mutation analysis was carried out for the LRP5 domain known to cause high bone mass. Results: Biochemical evaluation excluded most secondary causes of dense bones, and male-to-male transmission in her family indicated autosomal dominant inheritance. PCR amplification and sequencing of LRP5 exons 2-4 and adjacent splice sites revealed heterozygosity for a new LRP5 missense mutation, Arg154Met. Conclusions: LRP5 Arg154Met is a novel defect that changes the same first ",-propeller" module as the eight previously reported LRP5 gain-of-function missense mutations. Arg154Met alters a region important for LRP5 antagonism by dickkopf (Dkk). Therefore, our patient's extensive oropharyngeal exostoses and endosteal hyperostosis likely reflect increased Wnt signaling and show that exuberant LRP5 effects are not always benign. [source] A cyclic-di-GMP receptor required for bacterial exopolysaccharide productionMOLECULAR MICROBIOLOGY, Issue 6 2007Vincent T. Lee Summary Bis-(3,,5,)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide. [source] A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli,Expression and characterization of cytochrome-tagged Complex I subunitsPROTEIN SCIENCE, Issue 8 2010Tobias Gustavsson Abstract Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c550. Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c550 domain in all the fusion proteins exhibited normal spectra and redox properties, with an Em of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c -tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins. [source] Chemical synthesis and biotinylation of the thrombospondin domain TSR2PROTEIN SCIENCE, Issue 5 2009Theresa K. Tiefenbrunn Abstract The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site-specific labeling and detailed structure-function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In addition to the natural domain, five sites for side chain modification were evaluated and two were found to be compatible with oxidative folding. Several challenges were encountered during peptide synthesis due to the functional complexity of the domain. These challenges were overcome by the use of new solid supports, scavengers, and the testing of multiple ligation sites. We also describe an unusual sequence-specific protecting group migration observed during cleavage resulting in +90 Da and +194 Da adducts. Synthetic access to this domain enables the synthesis of a number of variants that can be used to further our understanding of the biochemical interaction network of thrombospondin and provide insight into the structure and function of this important antitumorogenic protein domain. [source] Solution structure and proposed domain,domain recognition interface of an acyl carrier protein domain from a modular polyketide synthasePROTEIN SCIENCE, Issue 10 2007Viktor Y. Alekseyev Abstract Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain,domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain,domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14,94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 ± 0.09 Å for the backbone atoms (1.21 ± 0.13 Å for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 ± 0.10 Å (0.99 ± 0.11 Å for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein,protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition. [source] Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domainPROTEIN SCIENCE, Issue 8 2005Charmaine Hoang Abstract The pseudouridine synthase TruB is responsible for the universally conserved post-transcriptional modification of residue 55 of elongator tRNAs. In addition to the active site, the "thumb," a peripheral domain unique to the TruB family of enzymes, makes extensive interactions with the substrate. To coordinate RNA binding and release with catalysis, the thumb may be able to sense progress of the reaction in the active site. To establish whether there is a structural correlate of communication between the active site and the RNA-sequestering thumb, we have solved the structure of a catalytically inactive point mutant of TruB in complex with a substrate RNA, and compared it to the previously determined structure of an active TruB bound to a reaction product. Superposition of the two structures shows that they are extremely similar, except in the active site and, intriguingly, in the relative position of the thumb. Because the two structures were solved using isomorphous crystals, and because the thumb is very well ordered in both structures, the displacement of the thumb we observe likely reflects preferential propagation of active site perturbations to this RNA-binding domain. One of the interactions between the active site and the thumb involves an active site residue whose hydrogen-bonding status changes during the reaction. This may allow the peripheral RNA-binding domain to monitor progress of the pseudouridylation reaction. [source] Arm,domain interactions can provide high binding cooperativityPROTEIN SCIENCE, Issue 10 2004Robert Schleif Abstract Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology. A well-studied example of this type of protein,protein interaction occurs between the yeast homeodomain proteins, MAT ,2 and MAT a1, which form a high-affinity heterodimer on DNA. The carboxyl-terminal arm extending from MAT ,2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1. Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change. This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase. The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions. [source] Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in ArabidopsisTHE PLANT JOURNAL, Issue 2 2003Yafan Huang Summary CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1,8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1,8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway. [source] High levels of intra-specific variation in the NG repeat region of the Pinctada maxima N66 organic matrix proteinAQUACULTURE RESEARCH, Issue 9 2009Carolyn Smith-Keune Abstract There has been wide speculation about the functional importance, and potential contribution to the varying nacre characteristics of pearl oysters, of variation in the length of an asparagine,glycine-rich repeat domain (the NG region) within the key organic matrix proteins nacrein and N66. This speculation has centred on large inter-species differences in the length of the NG protein domain. We report for the first time the presence of significant intra-specific (allelic) polymorphism in both the length and the amino acid sequence of the NG repeat domain of N66 within both wild and hatchery populations of Pinctada maxima. We also detected the presence of a second putative N66-like locus within the P. maxima genome. Confirmation of gene expression of the various alleles at the two loci identified in P. maxima is now required, together with experimental testing of functional differences in the carbonic anhydrase activity of the alleles identified. [source] Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Tat-Cheung Cheng Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16,197 (DBE16,197) was produced for structural determination. In this report, the crystallization and preliminary X-ray diffraction data analysis of the DBE16,197 protein domain are described. Crystals of DBE16,197 were grown by the sitting-drop vapour-diffusion method at 289,K using ammonium phosphate as a precipitant. The crystals belonged to space group P212121. Data were collected that extended to beyond 2,Å resolution. [source] A birth-to-death view of mRNA from the RNA recognition motif perspective,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2008Terri Goss Kinzy Abstract RNA binding proteins are a large and varied group of factors that are the driving force behind post-transcriptional gene regulation. By analogy with transcription factors, RNA binding proteins bind to various regions of the mRNAs that they regulate, usually upstream or downstream from the coding region, and modulate one of the five major processes in mRNA metabolism: splicing, polyadenylation, export, translation and decay. The most abundant RNA binding protein domain is called the RNA Recognition Motif (RRM)1. It is probably safe to say that an RRM-containing protein is making some contact with an mRNA throughout its existence. The transcriptional counterpart would likely be the histones, yet the multitude of specific functions that are results of RRM based interactions belies the universality of the motif. This complex and diverse application of a single protein motif was used as the basis to develop an advanced graduate level seminar course in RNA:protein interactions. The course, utilizing a learner-centered empowerment model, was developed to dissect each step in RNA metabolism from the perspective of an RRM containing protein. This provided a framework to discuss the development of specificity for the RRM for each required process. [source] Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesisBIOPOLYMERS, Issue 9 2010Mary Moon Abstract Nikkomycin Z is a dipeptide antifungal antibiotic characterized by two nonproteinogenic amino acids, nikkomycin CZ and 4-(4,-hydroxy-2,-pyridinyl)-homothreonine (HPHT). The HPHT scaffold is assembled by an aldol reaction between 2-oxobutyrate and picolinaldehyde, the latter of which is derived from picolinic acid that is activated and loaded to coenzyme A by the aryl-activating adenylation enzyme, NikE. We now provide evidence that NikE is also involved in the activation and loading of the ,-keto acid precursor, 4-(2,-pyridinyl)-2-oxo-4-hydroxyisovalerate (POHIV), to a phosphopantetheinyl group of an acyl carrier protein domain of NikT. POHIV was synthesized using Escherichia coli 2-dehydro-3-deoxy-phosphogluconate aldolase, and phenylalanine dehydrogenase from Bacillus sp. NRRL B-14911 was used to prepare the ,-amino acid, 4-(2,-pyridinyl)-homothreonine (PHT). Using the carboxylic acid-dependent, ATP-[32P]PPi exchange assay, NikE is shown to activate both picolinic acid and POHIV but not PHT. Furthermore, NikE loads POHIV to holo-NikT to generate a new thioester-linked intermediate, which was not observed using a NikT(S33A) mutant. Thus, NikE activates two distinct carboxylic acids to form two new thioester intermediates, one of which is subsequently reduced to the aldehyde and the other that likely serves as a substrate for the aminotransferase domain of NikT prior to condensation with nikkomycin CZ to yield the dipeptide. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 791,801, 2010. [source] Evolutionary and functional analysis of the tailless enhancer in Musca domestica and Drosophila melanogasterEVOLUTION AND DEVELOPMENT, Issue 1 2006Naomi S. Wratten SUMMARY To further understand the evolutionary dynamics of the regulatory interactions underlying development, we expand on our previous analysis of hunchback and compare the structure and function of the tailless enhancer between Musca domestica and Drosophila melanogaster. Our analysis shows that although the expression patterns and functional protein domains of tll are conserved between Musca and Drosophila, the enhancer sequences are unalignable. Upon closer investigation, we find that these highly diverged enhancer sequences encode the same regulatory information necessary for Bicoid, Dorsal, and the terminal system to drive tll expression. The binding sites for these transcription factors differ in the sequence, number, spacing, and position between the Drosophila and Musca tll enhancers, and we were unable to establish homology between binding sites from each species. This implies that the Musca and Drosophila Bcd-binding sites have evolved de novo in the 100 million years since these species diverged. However, in transgenic Drosophila embryos the Musca tll enhancer is able to drive the same expression pattern as endogenous Drosophila tll. Therefore, during the rapid evolution of enhancer sequences individual binding sites are continually lost and gained, but the transcriptional output is maintained by compensatory mutations in cis and in trans. [source] Identification of an osteopontin-like protein in fish associated with mineral formationFEBS JOURNAL, Issue 17 2007Vera G. Fonseca Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates. [source] Structural consequences of site-directed mutagenesis in flexible protein domainsFEBS JOURNAL, Issue 8 200156)S mutant of RhoGDI, NMR characterization of the L(5 The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45,56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein. [source] The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acidsFEBS JOURNAL, Issue 8 2000Chromosomal localization of the BSM1 gene, porcine counterparts, relations to ovine We previously elucidated five distinct protein domains (I,V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of ,,55 tandem repeats of 329 amino acids and that domains III,V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2.2,q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92,94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68,94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes. [source] Listeriolysin O: a key protein of Listeria monocytogenes with multiple functionsFEMS MICROBIOLOGY REVIEWS, Issue 4 2006Samer Kayal Abstract Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure,function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed. [source] ALys amyloidosis caused by compound heterozygosity in Exon 2 (Thr70Asn) and Exon 4 (Trp112Arg) of the lysozyme gene,,HUMAN MUTATION, Issue 1 2006Christoph Röcken Abstract Hereditary amyloidoses are caused by germline mutations, which increase the propensity of a protein to form cross-, aggregates and deposit as amyloid. Hereditary amyloidoses are particularly interesting as they help to understand how changes in the primary structure of an otherwise non-amyloidogenic protein contribute to amyloidogenesis. Here we report on a novel form of systemic ALys amyloidosis, caused by compound heterozygosity in exon 2 (p.T70N) and exon 4 (p.W112R) of the lysozyme gene (LYZ), with both mutations being present on the same allele. This type of hereditary ALys amyloidosis is characterized by extended amyloid deposits in the upper gastrointestinal tract, entire colon, and kidney, leading to gastrointestinal bleeding. Both mutations are probably effective in disease manifestation. The novel mutation at position 112 in the mature protein is located within the ,-helical domain of the protein and therefore outside the cluster of residues that has so far been implicated in ALys amyloidosis. Taken together with the p.T70N mutation, this results in a lysozyme species where the correct folding of various protein domains is probably impaired and increases the propensity of amyloid fibril formation. Interestingly, this form of ALys amyloidosis is also characterized by the occurrence of proteolytic fragments of lysozyme in the amyloid deposits. © 2005 Wiley-Liss, Inc. [source] Genetic and clinical aspects of X-linked hydrocephalus (L1 disease): Mutations in the L1CAM geneHUMAN MUTATION, Issue 1 2001Sabine Weller Abstract L1 disease is a group of overlapping clinical phenotypes including X-linked hydrocephalus, MASA syndrome, spastic paraparesis type 1, and X-linked agenesis of corpus callosum. The patients are characterized by hydrocephalus, agenesis or hypoplasia of corpus callosum and corticospinal tracts, mental retardation, spastic paraplegia, and adducted thumbs. The responsible gene, L1CAM, encodes the L1 protein which is a member of the immunoglobulin superfamily of neuronal cell adhesion molecules. The L1 protein is expressed in neurons and Schwann cells and seems to be essential for nervous system development and function. The patients' gene mutations are distributed over the functional protein domains. The exact mechanisms by which these mutations cause a loss of L1 protein function are unknown. There appears to be a relationship between the patients' clinical phenotype and the genotype. Missense mutations in extracellular domains or mutations in cytoplasmic regions cause milder phenotypes than those leading to truncation in extracellular domains or to non-detectable L1 protein. Diagnosis of patients and carriers, including prenatal testing, is based on the characteristic clinical picture and DNA mutation analyses. At present, there is no therapy for the prevention or cure of patients' neurological disabilities. Hum Mutat 18:1,12, 2001. © 2001 Wiley-Liss, Inc. [source] Detection and analysis of alternative splicing in the silkworm by aligning expressed sequence tags with the genomic sequenceINSECT MOLECULAR BIOLOGY, Issue 2 2005X.-F. Zha Abstract We identified 277 alternative splice forms in silkworm genes based on aligning expressed sequence tags with genomic sequences, using a transcipt assembly program. A large fraction (74%) of these alternative splices are located in protein-coding regions and alter protein products, whereas only 26% are in untranslated regions. From the alternative splices located in protein-coding regions, some (43%) affect protein domains that bind various biological molecules. The vast majority of the detected alternative forms in this study appear to be novel, and potentially affect biologically meaningful control of function in silkworm genes. Our results indicate that alternative splicing in silkworm largely produces protein diversity and functional diversity, and is a widely used mechanism for regulating gene expression. [source] Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicinaINSECT MOLECULAR BIOLOGY, Issue 5 2003I. A. Hansen Abstract The process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta. We identified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast-two-hybrid-system. The receptor-binding domain of arylphorin was located within domain 3 of the arylphorin monomer. The ligand-binding domain of the hexamerin receptor was mapped to the extreme N-terminus of the receptor. The binding domains identified exhibit no similarity to any functional protein domains known to date. Additionally, we identified two previously unknown protein-interactors of the hexamerin receptor. The results of this study provide further insights regarding the mechanism of the receptor-mediated endocytosis of storage proteins in insects. [source] Extensive gene conversion between CCR2 and CCR5 in domestic cat (Felis catus)INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2007P. J. Esteves Summary Homogenization of the CC-motif chemokine receptors CCR2 and CCR5 of cat (Felis catus) is documented and shown to be the outcome of gene conversion within the feline lineage. All regions were concerned, except the three extracellular protein domains (N- and C-tails, and ECL2), suggesting that structural differentiation at these domains could be related to pathogen susceptibility. [source] Serum and glucocorticoid-regulated protein kinases: Variations on a themeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Maude Tessier Abstract The phosphatidylinositol 3, kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers,phosphatidylinositides that are phosphorylated at the 3, position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (,, ,, and ,; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3, phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Domain,ligand mapping for enzymesJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2010Matthew Bashton Abstract In this paper we provide an overview of our current knowledge of the mapping between small molecule ligands and protein domains. We give an overview of the present data resources available on the Web, which provide information about protein,ligand interactions, as well as discussing our own PROCOGNATE database. We present an update of ligand binding in large protein superfamilies and identify those ligands most frequently utilized by nature. Finally we discuss potential uses for this type of data. Copyright © 2009 John Wiley & Sons, Ltd. [source] Lipid binding region (2303,2332) is involved in aggregation of recombinant human FVIII (rFVIII),JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2005Karthik Ramani Abstract Factor VIII (FVIII) is a multi-domain protein that is important in the clotting cascade. Its deficiency causes Hemophilia A, a bleeding disorder. The unfolding of protein domains can lead to physical instability such as aggregation, and hinder their use in replacement therapy. It has been shown that the aggregation of rFVIIII is initiated by small fluctuations in the protein's tertiary structure (Grillo et al., 2001, Biochemistry 40:586,595). We have investigated the domain(s) involved in the initiation of aggregation using circular dichroism (CD), size exclusion chromatography (SEC), fluorescence anisotropy, domain specific antibody binding, and clotting activity studies. The studies indicated that aggregation may be initiated as a result of conformational change in the C2 domain encompassing the lipid-binding region (2303,2332). The presence of O -phospho- L -Serine (OPLS), which binds to the lipid-binding region of FVIII, prevented aggregation of the protein. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:1288,1299, 2005 [source] Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leavesPLANT BIOLOGY, Issue 2008N. Ay Abstract Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed. [source] | |||||||||||||||||||