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Complex Assembly (complex + assembly)
Selected AbstractsComplex assembly, crystallization and preliminary X-ray crystallographic studies of MHC H-2Kd complexed with an HBV-core nonapeptideACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Minghai Zhou In order to establish a system for structural studies of the murine class I major histocompatibility antigen complex (MHC) H-2Kd, a bacterial expression system and in vitro refolding preparation of the complex of H-2Kd with human ,2m and the immunodominant peptide SYVNTNMGL from hepatitis B virus (HBV) core-protein residues 87,95 was employed. The complex (45,kDa) was crystallized; the crystals belong to space group P2221, with unit-cell parameters a = 89.082, b = 110.398, c = 47.015,Å, , = , = , = 90°. The crystals contain one complex per asymmetric unit and diffract X-rays to at least 2.06,Å resolution. The structure has been solved by molecular replacement and is the first crystal structure of a peptide,H-2Kd complex. [source] Complex assembly, crystallization and preliminary X-ray crystallographic studies of duck MHC class I moleculeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Jianhua Zhang In order to understand the biological properties of the immune systems of waterfowl and to establish a system for structural studies of duck class I major histocompatibility complex (DuMHC I), a complex of DuMHC I with duck ,2 -microglobulin (Du,2m) and the peptide AEIEDLIF (AF8) derived from H5N1 NP residues 251,258 was assembled. The complex was crystallized; the crystals belonged to space group C2221, with unit-cell parameters a = 54.7, b = 72.4, c = 102.2,Å, and diffracted to 2.3,Å resolution. Matthews coefficient calculation and initial structure determination by molecular replacement showed that the crystals did not contain the whole DuMHC I complex, but instead contained the DuMHC I ,3 domain and a Du,2m molecule (DuMHC I ,3+,2m). Another complex of DuMHC I with the peptide IDWFDGKE derived from a chicken fusion protein also generated the same results. The stable structure of DuMHC I ,3+,2m may reflect some unique characteristics of DuMHC I and pave the way for novel MHC structure-related studies in the future. [source] Free-Standing All-Nanoparticle Thin Fibers: A Novel Nanostructure Bridging Zero- and One-Dimensional Nanoscale Features,ADVANCED MATERIALS, Issue 3 2009Jia Yan Zero-dimensional nanoparticle structures have a prominent role as building blocks for complex assemblies. However, major barriers to the construction of 1D assemblies using 0D elements exist. The fabrication of free-standing all-nanoparticles thin fibers is presented, where the fibers have a uniform diameter (1.5,µm) and a high aspect ratio (length/diameter , 2500). [source] Controlled Crystallization of Calcite Under Surface Electric Field Due to Polarized Hydroxyapatite CeramicsJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 7 2009Norio Wada We examined effects of surface electric fields for the crystallization of calcite on polarized hydroxyapatite ceramics with and without polyacrylic acid (PAA) as soluble additive. Both on negatively and positively charged surfaces without PAA, the only precipitates were rhombohedra calcite crystals with the face of the {10.4} plane favorably oriented parallel to the surfaces. This oriented growth was explained by the nucleation theory in the presence of an external electric field. However, the addition of PAA drastically changed the situation of the calcite crystals, i.e., the crystallites were the hemispheric aggregates of calcite needles with a facetted rhombohedral {10.4} end face and flat island-shaped aggregates of ones with a rough (00.1) end face having a triangular shape. The calcite needles grew along the crystallographic [00.1] axis. This oriented growth was explained by epitaxy on the PAA,Ca2+ complexes adsorbing on the surfaces. The morphology of the PAA,Ca2+ complex assemblies adsorbing on the surfaces before the calcite nucleation was an important factor to control the structure of calcite aggregates formed following. This morphology was controlled by properties of the surface electric field and the spatial distribution of the negatively and positively charged sites in the PAA,Ca2+ complexes. [source] Cloning of Xenopus orthologs of Ctf7/Eco1 acetyltransferase and initial characterization of XEco2FEBS JOURNAL, Issue 24 2008Masatoshi Takagi Sister chromatid cohesion is important for the correct alignment and segregation of chromosomes during cell division. Although the cohesin complex has been shown to play a physical role in holding sister chromatids together, its loading onto chromatin is not sufficient for the establishment of sister chromatid cohesion. The activity of the cohesin complex must be turned on by Ctf7/Eco1 acetyltransferase at the replication forks as the result of a specific mechanism. To dissect this mechanism in the well established in vitro system based on the use of Xenopus egg extracts, we cloned two Xenopus orthologs of Ctf7/Eco1 acetyltransferase, XEco1 and XEco2. Both proteins share a domain structure with known members of Ctf7/Eco1 family proteins. Moreover, biochemical analysis showed that XEco2 exhibited acetyltransferase activity. We raised a specific antibody against XEco2 and used it to further characterize XEco2. In tissue culture cells, XEco2 gradually accumulated in nuclei through the S phase. In nuclei formed in egg extract, XEco2 was loaded into the chromatin at a constant level in a manner sensitive to geminin, an inhibitor of the pre-replication complex assembly, but insensitive to aphidicolin, an inhibitor of DNA polymerases. In both systems, no specific localization was observed during mitosis. In XEco2-depleted egg extracts, DNA replication occurred with normal kinetics and efficiency, and the condensation and sister chromatid cohesion of subsequently formed mitotic chromosomes was unaffected. These observations will serve as a platform for elucidating the molecular function of Ctf7/Eco1 acetyltransferase in the establishment of sister chromatid cohesion in future studies, in which XEco1 and XEco2 should be dissected in parallel. [source] Identification and characterization of cytochrome bc1 subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc1 subunitsFEBS JOURNAL, Issue 17 2007Vincenzo Zara We have examined the status of the cytochrome bc1 complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc1 complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc1 complex was detected as a mixed population of enzymes, consisting of cytochrome bc1 dimers, and ternary complexes of cytochrome bc1 dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc1 dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc1 subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc1 subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c1 associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc1 subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc1 complex assembly. [source] NEMO oligomerization in the dynamic assembly of the I,B kinase core complexFEBS JOURNAL, Issue 10 2007Elisabeth Fontan NF-,B essential modulator (NEMO) plays an essential role in the nuclear factor ,B (NF-,B) pathway as a modulator of the two other subunits of the I,B kinase (IKK) complex, i.e. the protein kinases, IKK, and IKK,. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-,B stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1,. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKK, and IKK,, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKK, dimers are present that are less stable than IKK, dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKK,,NEMO and IKK,,NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKK,,IKK, heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKK, and IKK,. [source] A picture says more than a thousand words: Structural insights into hepatitis C virus translation initiation,HEPATOLOGY, Issue 6 2006Pantxika Bellecave Ph.D. Protein synthesis in mammalian cells requires initiation factor eIF3, a ,750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5,-cap or an internal ribosome entry site (IRES). Cryoelectron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5,-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly. [source] Role of the transmembrane domain of glycoprotein IX in assembly of the glycoprotein Ib,IX complexJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007S.-Z. LUO Summary.,Background:,The glycoprotein (GP) Ib,IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIb, and GPIb, subunits interact and participate in complex assembly. Objectives and methods:,Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb,IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results:,Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb,IX complex. In contrast, replacing the GPIX TM domain (residues 132,153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIb,, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions:,The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb,IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex. [source] AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture openingMOLECULAR MICROBIOLOGY, Issue 6 2010Jon W. Weeks Summary In Escherichia coli, the TolC,AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four-residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC-mediated phage or colicin infection. However, despite the null-like drug sensitivity phenotype, chemical cross-linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA-mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB-dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state. [source] Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaAMOLECULAR MICROBIOLOGY, Issue 5 2004Valorie T. Ryan Summary Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony. [source] Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2000B. Gopal N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis. It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors. NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P212121, with unit-cell parameters a = 46.6, b = 64.2, c = 90.1,Å. A native data set complete to 1.6,Å resolution has been collected from a single crystal. [source] Lighting up gap junction channels in a flashBIOESSAYS, Issue 10 2002W. Howard Evans Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half-lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells by high-resolution time-lapse microscopy. With some studies, however, there are potential short-comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels ,old' and ,new' connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events.1 BioEssays 24:876,880, 2002. © 2002 Wiley Periodicals, Inc. [source] Lipid,nucleic acids interactions as base for organization and expression of cellular genomeINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 1 2010V. V. Kuvichkin Abstract Although lipid,nucleic acid interactions have been studied, with certain or little progress, for more than 30 years, it is only in recent years that the problem has received particular attention. It should, however, be noted that most studies deal with DNA-cationic surfactants interactions, whereas DNA-zwitterionic interactions, which are more complex and close to nature, are poorly investigated. The long-standing studies of the triple complexes: DNA,phosphatidylcholine liposomes,divalent metal cations allow us to confirm that these complexes are responsible for the formation of not only the structures existing in DNA,cationic liposome complexes but also some other cellular structures. The author proposed hypothesis about the involvement of direct DNA,lipid interactions in the nuclear pore assembly. Only taking into account interactions between DNA and lipids of cellular membrane, one can explain the origin of such structures as nucleoid, nuclear pore, and nuclear matrix. The formation of triple complexes was accompanied by the aggregation and partial fusion of liposomes as was shown by cryo-TEM technique. The author has presented new data on the structure of triple complexes, which were obtained by phase contrast cryo-TEM. Biophysical data on the liposomes fusion during triple complex formation and perspective of their computer simulation are also presented. DNA acts as a fusogen in this process and it unwinds in the region of liposomes fusion. The nuclear envelope and pore complexes assembly is provided by membrane vesicles fusion. Author has proposed that the DNA-induced fusion of zwitterionic liposomes in vitro may suggest the involvement of direct lipids,DNA interaction in nuclear envelope assembly. © 2009 Wiley Periodicals, Inc. 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