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Structural Domains (structural + domain)
Selected AbstractsTriple-Decker Transition-Metal Complexes (CnHn)M(B6C)M(CnHn) (M = Fe, Ru, Mn, Re; n = 5, 6) Containing Planar Hexacoordinate Carbon AtomsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 13 2006Si-Dian Li Abstract A density functional theory investigation is presented in this work on a new class of triple-decker complexes (CnHn)M(B6X)M(CnHn) (M = Fe, Ru, Mn, Re; X = B, C, N; n = 5, 6) containing almost perfect planar hexacoordinate carbon atoms and other planar hexacoordinate nonmetals at the centers of the B6X middle-deckers. Effective d,, coordination interactions between the partially filled 3d orbitals of the transition-metal center and the delocalized , orbitals of the three parallel ligands maintain the stabilities of these triple-decker complexes. The strong IR absorption peaks of (CnHn)M(B6X)M(CnHn) complexes mainly originate from the in-plane and off-plane vibrations of their planar hexacoordinate nonmetal centers. The results obtained in this work provide a possible new approach to characterize planar hexacoordinate carbon-containing systems in future experiments and expand the structural domain of transition-metal complexes by introducing inorganic B6X middle-deckers into traditional sandwich-type structures. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Physical characterization of plakophilin 1 reconstituted with and without zincFEBS JOURNAL, Issue 14 2000Ilse Hofmann Plakophilin 1 (PKP1) belongs to the arm -repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The , helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 ± 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20,w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 × 10,7 cm2·s,1. The molecular mass of 2.44 × 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation. [source] Leichhardt's maps: 100 years of change in vegetation structure in inland QueenslandJOURNAL OF BIOGEOGRAPHY, Issue 1 2008R. J. Fensham Abstract Aim, To address the hypothesis that there has been a substantial increase in woody vegetation cover (,vegetation thickening') during the 100 years after the burning practices of aboriginal hunter-gatherers were abruptly replaced by the management activities associated with pastoralism in north-east Australia. Location, Three hundred and eighty-three sites on 3000 km transect, inland Queensland, Australia. Methods, Vegetation structure descriptions from the route notes of the first European exploration of the location by Ludwig Leichhardt in 1844,45 were georeferenced and compiled. Leichhardt's application of structural descriptors (e.g. ,scrub', ,open forest', ,plain') was interpreted as domains within a matrix of tall stratum and low stratum woody cover. Woody cover was also interpreted for the same locations using aerial photography that largely pre-dates extensive land clearing (1940s,1970s) and compared with their structural domain in 1844,45. The fire-sensitive tree, cypress-pine (Callitris glaucophylla) was singled out for case study because it has been widely proposed that the density of this tree has substantially increased under European pastoral management. Results, The coarse resolution of this analysis indicates that the structure of the vegetation has been stable over the first 100 years of pastoralism. For example treeless or sparsely treed plains described by Leichhardt (1844,45) had the same character on the aerial photography (1945,78). Leichhardt typically described vegetation that includes cypress-pine as having a ,thicket' structure suggesting dense regenerating stands of small trees, consistent with the signature typical on the aerial photography. Main conclusions, A large data set of geographically located descriptions of vegetation structure from the first European traverse of inland Australia compared with vegetation structure determined from aerial photography does not support the hypothesis that vegetation thickening has been extensive and substantial. On the contrary the study suggests that the structure of the vegetation has been relatively stable for the first 100 years of European settlement and pastoralism except for those areas that have been affected by broad-scale clearing. [source] Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio choleraeMOLECULAR MICROBIOLOGY, Issue 4 2000Thomas J. Kirn The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus,pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres. [source] Role of the N-terminal Region in the Function of the Photosynthetic Bacterium Transcription Regulator PpsR,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Yoichi Yamazaki PpsR is a transcription repressor for the gene cluster encoding photosystem genes in Rhodobacter sphaeroides. Repression activity is accomplished by DNA binding on the promoter regions of the photosystem gene clusters, and depends on both the redox potential and the presence of antirepressor protein AppA. To understand DNA repression regulation by PpsR, we investigated the function of PpsR domains in self-association for DNA binding. We constructed domain-deletion mutants and verified DNA-binding activity and dimer formation. Gel shift assay for measuring the DNA-binding activity of three sequential N-terminal deletion mutants revealed that N-terminal deletions (of minimum 121 residues) caused loss of binding activity. Size-exclusion gel chromatography revealed that deletion mutant which lacks the N-terminal 121-amino acid deletion mutant to exist as a dimer, although it was less stable than the intact PpsR. The mutants lacking the adjacent regions, Q-linker region and the first Per-Ant-Sim domain, did not form dimers, suggesting the involvement of the N-terminal region in dimer formation. This region is thus considered to be a functional domain in self-association, although not yet identified as a structural domain. Circular dichroism spectrum of the N-terminal region fragment exhibited a ,/, structure. We conclude that this region is a structural and functional domain, contributing to PpsR repression through dimer stabilization. [source] The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrinPROTEIN SCIENCE, Issue 9 2009Yuanli Song Abstract The bundling of the N-terminal, partial domain helix (Helix C,) of human erythroid ,-spectrin (,I) with the C-terminal, partial domain helices (Helices A, and B,) of erythroid ,-spectrin (,I) to give a spectrin pseudo structural domain (triple helical bundle A,B,C,) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern,Volmer quenching constants of Helix C, of ,I bound to Helices A, and B, of ,I, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and ,I mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C, was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder. [source] Computer-aided NMR assay for detecting natively folded structural domains,PROTEIN SCIENCE, Issue 4 2006Takayuki Hondoh Abstract Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space. [source] Solution structure of the RWD domain of the mouse GCN2 proteinPROTEIN SCIENCE, Issue 8 2004Nobukazu Nameki Abstract GCN2 is the ,-subunit of the only translation initiation factor (eIF2,) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an , + , sandwich fold consisting of two layers: a four-stranded antiparallel ,-sheet, and three side-by-side ,-helices, with an ,,,,,,, topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive ,-turns that overlap with each other by two residues (triple ,-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in ,-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting ,-strand 4 and ,-helix 3. The structural architecture, including the triple ,-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins. [source] Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg.FEBS JOURNAL, Issue 12 2006Eleftherios Touloupakis A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 Å resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an ,,+ , N-terminal domain (residues 4,190) and a mainly ,-helical C-terminal domain (residues 191,257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170. [source] Genetic characterization of new Dobrava hantavirus isolate from GreeceJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Kirill Nemirov Abstract The first complete genome sequence of Dobrava hantavirus isolated from yellow-necked mouse Apodemus flavicollis trapped in the northeastern Greece is described. The S, M, and L segments of the Greek isolate of Dobrava virus are 1673, 3635, and 6532 nucleotides (nt) long, respectively, and encode the nucleocapsid (N) protein of 429 amino acids (aa), glycoprotein precursor of 1135 aa, and the L protein of 2151 aa. N protein contains three cysteine residues conserved in all known hantaviruses, as well as structural domains responsible for the RNA binding and presumable interaction with the apoptosis enhancer Daxx. All cysteine residues and glycosylation sites that are conserved among G1G2 sequences of all hantaviruses species were also found in the Greek isolate. The L protein contains all the polymerase motifs and structural domains found in other hantavirus polymerases. Comparison of the Greek isolate of Dobrava virus with other hantaviruses showed the highest level of sequence homology with Dobrava virus isolate from Slovenia. Other hantaviruses carried by Murinae rodents (Saaremaa, Hantaan, Seoul, and Thailand viruses) were more divergent and hantaviruses carried by Arvicolinae or Sigmodontinae rodents showed the highest genetic diversity with the Greek isolate of Dobrava. The results of phylogenetic analyses confirmed these observations and showed a monophily of all the Dobrava virus strains that, in turn, shared more ancient ancestors first with Saaremaa virus and then with other Murinae-borne hantaviruses. J. Med. Virol. 69:408,416, 2003. © 2003 Wiley-Liss, Inc. [source] Developmental change and function of chondroitin sulfate deposited around cerebellar Purkinje cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2005Yumiko Shimazaki Abstract Chondroitin sulfate is a long sulfated polysaccharide with enormous structural heterogeneity that binds with various proteins, such as growth factors, in a structure-dependent manner. In this study, we analyzed the expression of chondroitin sulfate in the postnatally developing cerebellar cortex by using three monoclonal antibodies against chondroitin sulfate, MO-225, 2H6, and CS-56, which recognize different structural domains in this polysaccharide. During the first postnatal week, the patterns of immunohistochemical staining made by these antibodies were quite similar, and the molecular layer, the granule cell layer, and Bergmann glial fibers in the external granular layer were densely stained. After postnatal day 12 (P12), the expression of 2H6 epitopes was down-regulated in the molecular layer, and the expression of CS-56 epitopes in this layer was also reduced after P16. On the other hand, the strong expression of MO-225 epitopes, GlcA(2S),1,3GalNAc(6S) (D unit)-containing structures, remained until adulthood. These chondroitin sulfate epitopes were observed around Purkinje cells, including cell soma and dendrites. Detailed immunohistochemical analysis suggested that chondroitin sulfate was deposited between Purkinje cell surfaces and the processes of Bergmann glia. Furthermore, the amount of pleiotrophin, a heparin-binding growth factor, in the cultured cerebellar slices was remarkably diminished after treatment with chondroitinase ABC or D unit-rich chondroitin sulfate. With the previous findings that pleiotrophin binds to D unit-rich chondroitin sulfate, we suggest that the D-type structure is important for the signaling of pleiotrophin, which plays roles in Purkinje cell,Bergmann glia interaction, and that the structural changes of chondroitin sulfate regulate this signaling pathway. © 2005 Wiley-Liss, Inc. [source] The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2006M. VAN DE WOUWER Summary.,Background: Thrombomodulin (TM) is predominantly a vascular endothelial cell plasma membrane glycoprotein that, via distinct structural domains, interacts with multiple ligands, thereby modulating coagulation, fibrinolysis, complement activation, inflammation and cell proliferation. We previously reported that by mediating signals that interfere with mitogen-activated protein kinase and nuclear factor ,B pathways, the amino-terminal C-type lectin-like domain of TM has direct anti-inflammatory properties. Methods: In the current study, we use murine models of acute inflammatory arthritis and biochemical approaches to assess the mechanism by which the lectin-like domain of TM modifies disease progression. Results: Mice lacking the lectin-like domain of TM (TMLeD/LeDmice) develop inflammatory arthritis that is more rapid in onset and more severe than that developed in their wildtype counterparts. In two models of arthritis, treatment of mice with recombinant soluble lectin-like domain of TM significantly suppresses clinical evidence of disease and diminishes monocyte/macrophage infiltration into the synovium, with weaker expression of the pro-inflammatory high mobility group box chromosomal protein 1. While thrombin-TM mediated activation of thrombin activatable fibrinolysis inhibitor inactivates complement factors C3a and C5a, we show that TM has a second independent mechanism to regulate complement: the lectin-like domain of TM directly interferes with complement activation via the classical and lectin pathways. Conclusions: These data extend previous insights into the mechanisms by which TM modulates innate immunity, and highlight its potential as a therapeutic target for inflammatory diseases. [source] Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing fliesMEDICAL AND VETERINARY ENTOMOLOGY, Issue 1 2000A. C. Lessinger Summary This study reports the molecular characterization of the mtDNA control region (called the A + T-rich region in insects) of five dipteran species which cause myiasis: Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, Chrysomya megacephala Fabricius, Lucilia eximia Wiedemann (Diptera: Calliphoridae) and Dermatobia hominis Linnaeus Jr (Diptera: Oestridae). The control region in these species varies in length from 1000 to 1600 bp. Two structural domains with specific evolutionary patterns were identified. These were (1) conserved sequence blocks containing primary sequence motifs, including dinucleotide pyrimidine-purine series and long T-stretches, located at the 5, end adjacent to the tRNAIle gene and (2) a hypervariable domain at the 3, end characterized by increased nucleotide divergence and size variation. A high frequency of A,T transversions at nucleotide substitution level indicated directional mutation pressure. The phylogenetic usefulness of the insect control region is discussed. [source] Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domainsMOLECULAR MICROBIOLOGY, Issue 3 2007Yvonne Tourand Summary Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT164,449) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT1,163) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT164,449. Conversely, ResT1,164 may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain. [source] Computer-aided NMR assay for detecting natively folded structural domains,PROTEIN SCIENCE, Issue 4 2006Takayuki Hondoh Abstract Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space. [source] Automatic generation and evaluation of sparse protein signatures for families of protein structural domainsPROTEIN SCIENCE, Issue 1 2005Matthew J. Blades Abstract We identified key residues from the structural alignment of families of protein domains from SCOP which we represented in the form of sparse protein signatures. A signature-generating algorithm (SigGen) was developed and used to automatically identify key residues based on several structural and sequence-based criteria. The capacity of the signatures to detect related sequences from the SWISSPROT database was assessed by receiver operator characteristic (ROC) analysis and jack-knife testing. Test signatures for families from each of the main SCOP classes are described in relation to the quality of the structural alignments, the SigGen parameters used, and their diagnostic performance. We show that automatically generated signatures are potently diagnostic for their family (ROC50 scores typically >0.8), consistently outperform random signatures, and can identify sequence relationships in the "twilight zone" of protein sequence similarity (<40%). Signatures based on 15%,30% of alignment positions occurred most frequently among the best-performing signatures. When alignment quality is poor, sparser signatures perform better, whereas signatures generated from higher-quality alignments of fewer structures require more positions to be diagnostic. Our validation of signatures from the Globin family shows that when sequences from the structural alignment are removed and new signatures generated, the omitted sequences are still detected. The positions highlighted by the signature often correspond (alignment specificity >0.7) to the key positions in the original (non-jack-knifed) alignment. We discuss potential applications of sparse signatures in sequence annotation and homology modeling. [source] Protein transduction into human cells by adenovirus dodecahedron using WW domains as universal adaptorsTHE JOURNAL OF GENE MEDICINE, Issue 4 2006A. Garcel Abstract Background Direct protein transduction is a recent technique that involves use of peptide vectors. In this study, we demonstrate that adenovirus dodecahedron (Dd), a virus-like particle devoid of DNA and able to penetrate cells with high efficiency, can be used as a vector for protein delivery. Methods Taking advantage of Dd interaction with structural domains called WW, we have elaborated a universal adaptor to attach a protein of interest to this vector. Results A tandem of three WW structural domains derived from the Nedd4 protein enables the formation of stable complexes with Dd, without impairing its endocytosis efficiency. Our protein of interest fused to the triple WW linker is delivered by the dodecahedron in 100% of cells in culture with on average more than ten million molecules per cell. Conclusion These data demonstrate the great potential of adenovirus dodecahedron in combination with WW domains as a protein transduction vector. Copyright © 2006 John Wiley & Sons, Ltd. [source] SYMPOSIUM REVIEW: Revealing the structural basis of action of hERG potassium channel activators and blockersTHE JOURNAL OF PHYSIOLOGY, Issue 17 2010Matthew Perry Human ether-á-go-go related gene (hERG) potassium (K+) channels play a critical role in cardiac action potential repolarization. This is due, in large part, to the unique gating properties of these channels, which are characterized by relatively slow activation and an unusually fast and voltage-dependent inactivation. A large number of structurally diverse compounds bind to hERG and carry an unacceptably high risk of causing arrhythmias. On the other hand, drugs that increase hERG current may, at least in principle, prove useful for treatment of long QT syndrome. A few blockers have been shown to increase hERG current at potentials close to the threshold for channel activation , a process referred to as facilitation. More recently, a novel group of hERG channel activators have been identified that slow deactivation and/or attenuate inactivation. Structural determinants for the action of two different types of activators have been identified. These compounds bind at sites that are distinct from each other and also separate from the binding site of high affinity blockers. They reveal not only novel ways of chemically manipulating hERG channel function, but also interactions between structural domains that are critical to normal activation and inactivation gating. [source] The ,1 and ,6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kineticsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Janet L. Fisher The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The , family is the largest, with six different subtypes. The ,1 and ,6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the ,1 or ,6 subunit was expressed in mammalian cells along with ,3 and ,2L subunits and the kinetic properties examined with outside-out patch recordings. The ,1,3,2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the ,6,3,2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for ,6,3,2L, compared with the ,1,3,2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the ,1 and ,6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3,TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the ,1 and ,6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition. [source] Multiscale structure in sedimentary basinsBASIN RESEARCH, Issue 2 2004S. A. Stewart Hierarchies of superimposed structures are found in maps of geological horizons in sedimentary basins. Mapping based on three-dimensional (3D) seismic data includes structures that range in scale from tens of metres to hundreds of kilometres. Extraction of structures from these maps without a priori knowledge of scale and shape is analogous to pattern recognition problems that have been widely researched in disciplines outside of Geoscience. A number of these lessons are integrated and applied within a geological context here. We describe a method for generating multiscale representations from two-dimensional sections and 3D surfaces, and illustrate how superimposed geological structures can be topologically analysed. Multiscale analysis is done in two stages , generation of scale-space as a geometrical attribute, followed by identification of significant scale-space objects. Results indicate that Gaussian filtering is a more robust method than conventional moving average filtering for deriving multiscale geological structure. We introduce the concept of natural scales for identifying the most significant scales in a geological cross section. In three dimensions, scale-dependent structures are identified via an analogous process as discrete topological entities within a four-dimensional scale-space cube. Motivation for this work is to take advantage of the completeness of seismic data coverage to see ,beyond the outcrop' and yield multiscale geological structure. Applications include identifying artefacts, scale-specific features and large-scale structural domains, facilitating multiscale structural attribute mapping for reservoir characterisation, and a novel approach to fold structure classification. [source] Antimicrobial peptide RP-1 structure and interactions with anionic versus zwitterionic micellesBIOPOLYMERS, Issue 1 2009Sarah Bourbigot Abstract Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing ,-core structural domains. The C-terminal ,-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic ,-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 1,13, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | ||||||||||||||||