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Human papillomaviruses (HPVs) cause cellular hyper-proliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles. [ABSTRACT FROM AUTHOR]

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant. [ABSTRACT FROM AUTHOR]

We report a highly ordered intercalated hexagonal binary superlattice of hydrophilically functionalized singlewalled carbon nanotubes (p-SWNTs) and surfactant (C12E5) cylindrical micelles. When p-SWNTs (with a diameter slightly larger than that of the C12E5 cylinders) were added to the hexagonally packed C12E5 cylindrical-micellar system, p- SWNTs positioned themselves in such a way that the freevolume entropies for both p-SWNTs and C12E5 cylinders were maximized, thus resulting in the intercalated hexagonal binary superlattice. In this binary superlattice, a hexagonal array of p- SWNTs is embedded in a honeycomb lattice of C12E5 cylinders. The intercalated hexagonal binary superlattice can be highly aligned in one direction by an oscillatory shear field and remains aligned after the shear is removed. [ABSTRACT FROM AUTHOR]

In higher eukaryotes, one of the two arginyl-tRNA synthetases (ArgRSs) has evolved to have an extended N-terminal domain that plays a crucial role in protein synthesis and cell growth and in integration into the multisynthetase complex (MSC). Here, we report a crystal structure of the MSC subcomplex comprising ArgRS, glutaminyl-tRNA synthetase (GlnRS), and the auxiliary factor aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)/p43. In this complex, the N-terminal domain of ArgRS forms a long coiled-coil structure with the N-terminal helix of AIMP1 and anchors the C-terminal core of GlnRS, thereby playing a central role in assembly of the three components. Mutation of AIMP1 destabilized the N-terminal helix of ArgRS and abrogated its catalytic activity. Mutation of the N-terminal helix of ArgRS liberated GlnRS, which is known to control cell death. This ternary complex was further anchored to AIMP2/p38 through interaction with AIMP1. These findings demonstrate the importance of interactions between the N-terminal domains of ArgRS and AIMP1 for the catalytic and noncatalytic activities of ArgRS and for the assembly of the higher-order MSC protein complex. [ABSTRACT FROM AUTHOR]

Structural analysis of branched DNA molecules (BDM) is important as model systems for DNA junctions and also as building units for DNA assembly. Although there have been efforts to study the structures of BDM, label-free solution structures have not been well determined yet. Here, we used a combination of synchrotron-based experimental tools and computational simulation to study the global structures of label-free BDM in solution. Overall structures of 3-arm and 4-arm BDM were revealed as an asymmetric T(or Y)-shape and a distorted X-shape, respectively. The internal structures of the DNA double helix were shown to have a canonical B-form for both the BDM. We also reconstructed the thermal denaturation process of BDM by determining the transient global structures over a wide range of temperatures. The proposed high-resolution structures of BDM are expected to provide fundamental information for studies of the biological function of junction DNAs and DNA assembly. [ABSTRACT FROM AUTHOR]

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement- mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPOhyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FccR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUClast). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFcfusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach. [ABSTRACT FROM AUTHOR]

In this study, a grazing-incidence X-ray scattering (GIXS) formula was derived for lamellar structures formed in thin films supported on substrates, and a comprehensive numerical analysis was performed using the scattering formula. A quantitative analysis was conducted of lamellar structures formed in nanometre-scaled thin films of a brush polymer, poly[oxy( n-decylthiomethylenyl)ethylene], supported on silicon substrates, by GIXS measurements during cooling and subsequent heating, and data analysis was performed using the scattering formula. This analysis provided details (long period, sub-layers and their thicknesses, volume fraction, bristle paracrystal distortion factor, and orientation) of the lamellar structure with varying temperature that are not easily obtained using conventional techniques. Moreover, a molecular structure model and electron density profiles were established. [ABSTRACT FROM AUTHOR]

The bacterial envelope stress response senses stress signals in the extracytoplasmic compartment, and activates σE-dependent transcription by degrading its antisigma factor RseA. RseB, a binding partner of RseA, plays a pivotal role in regulating this response, but its molecular mechanism is not understood. We therefore determined the crystal structure of Escherichia coli RseB at a resolution of 2.4 Å. RseB is composed of two domains linked by a flexible linker and forms a loosely packed dimer with two grooves on each side. This structural feature is confirmed by small-angle scattering in solution. Analysis of the binding of various RseA mutants to RseB allowed us to identify the major RseB-binding motif in RseA. These data, coupled with analysis of small-angle scattering of the RseA/RseB complex in solution, leads us to propose that two RseAs bind to the grooves of the dimeric RseB by conserved residues. The implications for modulating proteolytic cleavage of RseA are discussed. [ABSTRACT FROM AUTHOR]

Small angle x-ray scattering (SAXS) was used to estimate the degree of polymerization of polymer-grafted carbon nanotubes (CNTs) synthesized using a ‘grafting from’ method. This analysis characterizes the grafted polymer chains without cleaving them from CNTs, and provides reliable data that can complement conventional methods such as thermogravimetric analysis or transmittance electron microscopy. Acrylonitrile was polymerized from the surface of the CNTs by using redox initiation to produce poly-acrylonitrile-grafted CNTs (PAN-CNTs). Polymerization time and the initiation rate were varied to control the degree of polymerization. Radius of gyration (Rg) of PAN-CNTs was determined using the Guinier plot obtained from SAXS solution analysis. The results showed consistent values according to the polymerization condition, up to a maximum Rg = 125.70 Å whereas that of pristine CNTs was 99.23 Å. The dispersibility of PAN-CNTs in N,N-dimethylformamide was tested using ultraviolet–visible-near infrared spectroscopy and was confirmed to increase as the degree of polymerization increased. This analysis will be helpful to estimate the degree of polymerization of any polymer-grafted CNTs synthesized using the ‘grafting from’ method and to fabricate polymer/CNT composite materials. [ABSTRACT FROM AUTHOR]

Quantitative, non-destructive grazing-incidence X-ray scattering and specular X-ray reflectivity analysis with synchrotron radiation sources, along with spectroscopic ellipsometry analysis, were successfully used to characterize a series of low dielectric constant, nanoporous SiOCH dielectric thin films with nanometre-scale thicknesses prepared by radio-frequency inductively coupled plasma chemical vapor deposition of bistrimethylsilylmethane precursor and oxygen gas at various flow rate ratios followed by annealing at 298, 473, 573 or 673 K. These analyses provided important information on the structures and properties of the nanoporous films. The average size of the nanopores generated in each film was 3.07 nm in radius or less, depending on the process conditions. The film electron densities ranged from 414 to 569 nm−3, the refractive indices ranged from 1.434 to 1.512 at 633 nm wavelength, and the porosities ranged from 16.1 to 38.9%. Collectively, the present findings show that SiOCH thin films of the type reported here are suitable for use as low dielectric constant interdielectric layer materials in the fabrication of advanced integrated circuits. [ABSTRACT FROM AUTHOR]

In this study, we derive a grazing-incidence X-ray scattering (GIXS) formula to analyze quantitatively GIXS patterns for molecularly stacked layer structures in substrate-supported nanoscale thin films. We apply this formula in the quantitative analysis of GIXS patterns obtained for S-docosanylcysteine thin films on silicon substrates with native oxide layers. This analysis successfully provides information on the structural parameters and orientation of the molecular layer stack developed in S-docosanylcysteine thin films. [ABSTRACT FROM AUTHOR]

In the present study, structural analyses using synchrotron grazing incidence X-ray scattering, specular reflectivity and ellipsometry were performed on thin films of two novel polynorbornene derivatives, chiral poly(norbornene acid methyl ester) and racemic poly(norbornene acid n-butyl ester), which are potential low dielectric constant materials for advanced microelectronic and display applications. These analyses provided important information on the structure, electron density gradient across the film thickness, chain orientation, refractive index and thermal expansion characteristics of the polymers in substrate-supported thin films. The structural characteristics and properties of the thin films depended on the tacticity of the polymer chain and were further influenced by the film thickness and thermal annealing history. [ABSTRACT FROM AUTHOR]

The crystallization behavior of microbially synthesized poly(3-hydroxybutyrate) was studied in detail using time-resolved small-angle X-ray scattering. This polyester was found to undergo primary crystallization as well as secondary crystallization. In the primary crystallization, the thicknesses of the lamellar crystals were sensitive to the crystallization temperature, but no thickening was observed throughout the entire crystallization at a given temperature. The thickness of the lamellar crystals in the polyester was always larger than that of the amorphous layers. Secondary crystallization favorably occurred during the later stage of isothermal crystallization in competition with the continuous primary crystallization, forming secondary crystals in amorphous regions, in particular in the amorphous layers between the primarily formed lamellar crystal stacks. Compared to the primarily formed lamellar crystals, the secondary crystals had short-range-ordered structures of smaller size, a broader size distribution, and a lower electron density. [ABSTRACT FROM AUTHOR]

The mechanism of thermal pore generation in organosilicate thin films loaded with a six-armed star-shaped poly(ε-caprolactone) porogen was quantitatively investigated by using in-situ grazing-incidence small-angle X-ray scattering and thermogravimetry. These analyses found that the blend components have a limited miscibility that depends on the composition; for porogen loadings up to only 20 wt%, molecularly miscible blend films were obtained. Even for the miscible blend films, heating the films produced a curing reaction of the precursor matrix component, leading to the phase separation of the porogen component. This phase separation was found to begin at 393 K for 10 wt% porogen loaded films and at 373 K for 20 wt% porogen loaded films, and to continue for temperatures up to 423 K. The porogen aggregates remained and were confined within the matrix film without any further growth or movement until complete thermal decomposition above 564 K. [ABSTRACT FROM AUTHOR]