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Abstract: NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-Å resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11–12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11–12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11–12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.
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Feb 2005
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Abstract: The cryocooling of protein crystals to temperatures of around 100 K drastically reduces X-ray-induced radiation damage. The majority of macromolecular data collection is therefore performed at 100 K, yielding diffraction data of higher resolution and allowing structure determination from much smaller crystals. However, at third-generation synchrotron sources radiation damage at 100 K still limits the useful data obtainable from a crystal. For data collection at 15 K, realised by the use of an open-flow helium cryostat, a further reduction of radiation damage is expected. However, no systematic studies have been undertaken so far. In this present study, a total of 54 data sets have been collected from holoferritin and insulin crystals at 15 and 90 K in order to identify the effect of the lower data-collection temperature on the radiation damage. It is shown that data collection at 15 K has only a small positive effect for insulin crystals, whereas for holoferritin crystals radiation damage is reduced by 23% compared with data collection at 90 K.
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Mar 2007
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O.
Copie
,
K.
Rode
,
R.
Mattana
,
M.
Bibes
,
V.
Cros
,
G.
Herranz
,
A.
Anane
,
R.
Ranchal
,
E.
Jacquet
,
K.
Bouzehouane
,
M-a.
Arrio
,
Peter
Bencok
,
N. B.
Brookes
,
F.
Petroff
,
A.
Barthelemy
Abstract: We report a study of Co-doped La0.37Sr0.63TiO3-delta thin films grown by pulsed laser deposition in various oxygen pressure conditions. X-ray absorption spectroscopy and magnetic circular dichroism measurements at the Co L-2,L-3 edges reveal that the cobalt mainly substitutes for the titanium and is in an ionic state. Nevertheless, in some films, indications of additional cobalt metallic impurities were found, suggesting that the intrinsic character of this magnetic system remains questionable.
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Oct 2009
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Abstract: Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (L-Ala-P) have determined by X-ray crystallography to resolutions of 2.1 and 1.47 Å, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is D-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of L-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.
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Apr 2008
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Abstract: We measured coherent x-ray diffraction (CXD) on zeolite microcrystals in order to gain information on internal density distribution and to learn more about the strain developed during the synthesis and attachment process on the substrate. From the distortion and asymmetry of the diffraction pattern on the (020) Bragg peak, the strain field distribution is estimated. We inverted the diffraction patterns from a less strained crystal to obtain the three-dimensional image of the shape and internal strain fields using the error reduction and hybrid input–output phase retrieval algorithms. We also show a few examples of characteristic distortion modes relevant to CXD of zeolites.
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Mar 2010
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Abstract: By cunningly diffracting X-rays twice from an exploding nanometre-scale sphere, holographic images can be made of a tiny system evolving at lightning speed. The technique could be used to picture atomic dynamics.
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Aug 2007
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Abstract: The self-assembly of PEGylated peptides containing a modified sequence from the amyloid beta peptide, FEK LVFF, has been studied in aqueous solution. PEG molar masses PEG1k, PEG2k, and PEG10k were used in the conjugates. It is shown that the three FFK LVFF-PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The beta-sheet secondary structure of the peptide is retained in the FFK LVFF fibril core. At sufficiently high concentrations, FEK LVFF-PEG1k and FEK LVFF-PEG2k form a nema tic phase, while PEG10k-FEK LVFF exhibits a hexagonal columnar phase. Simultaneous small angle neutron scattering/shear flow experiments were performed to study the shear flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without disruption of the FFK LVFF beta-sheet structure leading to characteristic peaks in the X-ray diffraction pattern and FTIR spectra. The stability of beta-sheet structures was also studied in blends of FFKLVFF-PEG conjugates with poly(acrylic acid) (PAA). While PEG crystallization is only observed up to 25% PAA content in the blends, the FFK LVFF beta-sheet structure is retained up to 75% PAA.
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Jan 2010
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Abstract: There has been great interest recently in peptide amphiphiles and block copolymers containing biomimetic peptide sequences due to applications in bionanotechnology. We investigate the self-assembly of the peptide-PEG amphiphile FFFF-PEG5000 containing the hydrophobic sequence of four phenylalanine residues conjugated to PEG of molar mass 5000. This serves as a simple model peptide amphiphile. At very low concentration, association of hydrophobic aromatic phenylalanine residues occurs, as revealed by circular dichroism and UV/vis fluorescence experiments. A critical aggregation concentration associated with the formation of hydrophobic domains is determined through pyrene fluorescence assays. At higher concentration, defined ?-sheets develop as revealed by FTIR spectroscopy and X-ray diffraction. Transmission electron microscopy reveals self-assembled straight fibril structures. These are much shorter than those observed for amyloid peptides, the finite length may be set by the end cap energy due to the hydrophobicity of phenylalanine. The combination of these techniques points to different aggregation processes depending on concentration. Hydrophobic association into irregular aggregates occurs at low concentration, well-developed ?-sheets only developing at higher concentration. Drying of FFFF-PEG5000 solutions leads to crystallization of PEG, as confirmed by polarized optical microscopy (POM), FTIR and X-ray diffraction (XRD). PEG crystallization does not disrupt local ?-sheet structure (as indicated by FTIR and XRD). However on longer lengthscales the ?-sheet fibrillar structure is perturbed because spherulites from PEG crystallization are observed by POM.
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May 2009
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Abstract: The crystal structure of VEGF-E was solved by the sulfur single-wavelength anomalous dispersion method (S-SAD) using highly redundant low-resolution data collected at a wavelength of 1.7 Å with an estimated anomalous signal of 1.5%. 11 sulfur sites, nine out of 16 disulfide bonds and two out of 12 methionines could be located in the asymmetric unit using data truncated at a resolution of 4.1 Å; however, none of the common diffraction data-quality indicators for SAD allowed clear discrimination between successful and unsuccessful resolution cutoffs. The high solvent content of 75% allowed efficient density modification to be performed and an unbiased electron-density map of good quality to be generated. This study demonstrates the strength of S-¬SAD for phasing using accurate highly redundant data at low resolution.
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Nov 2006
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Abstract: New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
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Jan 2009
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