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Abstract: Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H+ binding. To initiate transport, binding of sugar and/or an H+ electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.
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Sep 2007
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Abstract: Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.
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Jan 2009
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Abstract: The biological role of manganese (Mn2+) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage {phi}6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn2+ by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn2+-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn2+, reduced RNA binding and a compromised elongation rate. Loss of Mn2+ binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn2+ coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn2+ is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.
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Oct 2008
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Abstract: We use optical-pump terahertz-probe spectroscopy to investigate the near-threshold behavior of the photoinduced insulator-to-metal (IM) transition in vanadium dioxide thin films. Upon approaching Tc a reduction in the fluence required to drive the IM transition is observed, consistent with a softening of the insulating state due to an increasing metallic volume fraction (below the percolation limit). This phase coexistence facilitates the growth of a homogeneous metallic conducting phase following superheating via photoexcitation. A simple dynamic model using Bruggeman effective medium theory describes the observed initial condition sensitivity.
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Nov 2007
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Abstract: Context. Silicate dust grains exist in a wide range of astronomical environments and understanding the effect of these on grain structure is of great interest, particularly the effect of thermal annealing on amorphous silicates as a possible route to the formation of crystalline grains. Although laboratory simulations have largely focussed on IR-spectroscopic measurements, since these relate directly to observational data, laboratory synchrotron techniques such as X-ray absorption spectroscopy (XAS) and X-ray scattering are becoming increasingly routine in the analysis of recovered materials. With the increasing prospect of performing astronomical XAS observations, there is much to be gained from applying these techniques to laboratory analogues.
Aims. Diagnostic markers for medium-range order and the effect of thermal annealing on these in amorphous silicates of differing Mg content are characterised using synchrotron X-ray methods.
Methods. Three synthetic amorphous silicates with high, medium and low Mg:Si ratios were annealed at varying temperatures up to 1300 K. X-ray Absorption Near Edge Structure (XANES) spectra at the Si and Mg K-shell absorption edges and X-ray scattering patterns for low values of the X-ray scattering wavevector were recorded for these along with comparative data for commercially produced amorphous SiO2 and mineral samples of forsterite, enstatite and quartz.
Results. XANES features due to short- and medium-range structure are identified at both Mg and Si edges and a new temperature dependent behaviour observed in the medium-range structure surrounding Mg and Si. Based on changes to the morphological details of the XANES spectra, the medium-range structure changes between an enstatite-like and forsterite-like coordination with increasing temperature and appears to correlate directly with Mg content. Low wavevector X-ray scattering features were also found to be diagnostic of the type of medium-range structural ordering. However, these features depend on whether the relative arrangement of clusters of medium-range structure exhibit semi-periodic ordering over the longer-range, which can vary with annealing.
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Jun 2008
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Abstract: We show that double-resonance spectra recorded during the simultaneous absorption of x-ray and microwave (MW) photons are a fingerprint of the perturbed electronic configuration of atomic species driven to ferromagnetic resonance. X-ray absorption measurements performed as a function of x-ray energy and polarization over the Fe?L 2,3 edges of single-crystal yttrium-iron garnet reveal MW-induced multiplet features related to angular momentum transfer from the MW field to localized Fe?3d magnetic sublevels. O?K -edge absorption spectra demonstrate the formation of dynamic 2p -orbital magnetization components at O sites coupled to the Fe magnetic moments at tetrahedral and octahedral sites. These results are compared with double-resonance x-ray absorption spectra of Permalloy, showing that the MW transition probability is distributed according to the hybridization character of the 3d states and proportional to the unperturbed unoccupied magnetic density of states of metals and insulators.
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Jun 2009
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Abstract: By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 °C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 °C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F680/F655 fluorescence intensity ratio. In contrast, heat treatment at 60 °C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F655), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F680 originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600–7500 μmol photons m−2 s−1) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 °C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.
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Jun 2007
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Abstract: The partition coefficients of potassium, DK, between molten sanidine, KAlSi3O8, and molten roedderite, K2Mg5Si12O30, with FeS-rich alloy and pure Fe metal liquids have been investigated in a multi-anvil press, between 5 and 15 GPa, at a temperature of 2173 K, and at an oxygen fugacity between 0.5 and 3 log units below the iron-wüstite (IW) buffer. No pressure dependence of the DK coefficients in sulphur-free and sulphur-bearing systems was found within the investigated pressure range. We also observed minor effect of the silicate melt composition for an nbo/t (non-bridging oxygen to tetrahedral cation ratio) higher than 0.8 ± 0.4. In contrast, the partitioning of potassium varies strongly with the metallic phase composition, with an increase of K-solubility in the metallic liquid for high sulphur and oxygen contents.
We review all available high-pressure data to obtain reliable DK coefficients for the interaction between molten silicates and Fe-alloy liquids at pressures and temperatures relevant to those of core formation in a terrestrial magma ocean. The dominant controlling parameters appear to be the temperature and the chemical composition of the metallic phase, with DK coefficients significantly increased with temperature, and with the sulphur and oxygen contents of the Fe-alloy liquid. Our considerations distinguish two extreme cases, with an S-free or S-bearing iron core, which yield K contents of ∼25 or ∼250 ppm, respectively. These two extreme values have very different consequences for thermal budget models of the Earth's core since its formation.
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Jan 2007
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Abstract: The development of the capability to engineer the surface properties of materials to match specific requirements demands high quality surface characterization techniques. The ideal tool should provide chemically specific structural characterization as well as surface sensitivity and depth profiling. Ideally the characterization method should also be applicable to systems both with and without long range order. X-ray absorption spectroscopy fine structure, when using the standard transmission detection system, provides all this information with the significant exception of surface sensitivity. In contrast, by detecting the reflected instead of the transmitted beam, it encompasses all these requirements because when the incident beam impinges onto a sample surface at glancing angles, in conditions close to the total reflection, only the outermost regions of the system under study are sampled. Such a technique provides information about the local structure as a function of depth as well as thin layer structure in the case of layered samples. Although it is potentially the ideal tool to study surface modified materials, experimental difficulties have hampered its widespread use in the fields of surface and materials sciences. As a solution to the experimental challenges, we provide a detailed description of an appropriate experimental station, the sample requirements, the measuring protocols, and software routines needed to optimize the collection of the data. To illustrate the capabilities of the technique the results obtained for a model multilayer sample are presented and analyzed under the total external reflection approximation.
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Jan 2007
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Abstract: LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.
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Jan 2009
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