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13 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	A multi-pronged approach to understanding the form and function of hStaufen protein Silvia Visentin , Giuseppe Cannone , James Doutch , Gemma Harris , Michael L. Gleghorn , Luke Clifton , Brian O. Smith , Laura Spagnolo Rna DOI: 10.1261/rna.072595.119 Diamond Proposal Number(s): [21035] Show Abstract ▼ Abstract: Staufen is a dsRNA binding protein that plays an essential role in many aspects of RNA regulation, such as mRNA transport, Staufen-mediated mRNA decay and the regulation of mRNA translation. Staufen is a modular protein characterized by the presence of conserved consensus amino acid sequences that fold into double-stranded RNA binding domains (RBDs) as well as degenerated RBDs that maintain the α-β-β-β-α fold but are unable to bind RNA and are instead involved in protein-protein interactions. The variety of biological processes in which Staufen participates in the cell suggests that this protein associates with many diverse RNA targets, some of which have been identified experimentally. Staufen binding mediates the recruitment of effectors via protein-protein and protein-RNA interactions. The structural determinants of a number of these interactions, as well as the structure of full-length Staufen, remain unknown. Here, we present the first solution structure models for full-length human Staufen155, showing that its domains are arranged as beads-on-a-string in the absence of RNA.	Dec 2019
	An introduction to classical molecular dynamics simulation for experimental scattering users Andrew R. Mccluskey , James Grant , Adam R. Symington , Tim Snow , James Doutch , Benjamin J. Morgan , Stephen C. Parker , Karen J. Edler Journal Of Applied Crystallography 52, 665-668 DOI: 10.1107/S1600576719004333 Open Access Show Abstract ▼ Abstract: Classical molecular dynamics simulations are a common component of multi-modal analyses of scattering measurements, such as small-angle scattering and diffraction. Users of these experimental techniques often have no formal training in the theory and practice of molecular dynamics simulation, leading to the possibility of these simulations being treated as a `black box' analysis technique. This article describes an open educational resource (OER) designed to introduce classical molecular dynamics to users of scattering methods. This resource is available as a series of interactive web pages, which can be easily accessed by students, and as an open-source software repository, which can be freely copied, modified and redistributed by educators. The topics covered in this OER include classical atomistic modelling, parameterizing interatomic potentials, molecular dynamics simulations, typical sources of error and some of the approaches to using simulations in the analysis of scattering data.	Jun 2019
B21-High Throughput SAXS	X-ray structure of full-length human RuvB-Like 2 – mechanistic insights into coupling between ATP binding and mechanical action Sara Silva , Jose A. Brito , Rocío Arranz , Carlos Óscar S. Sorzano , Christine Ebel , James Doutch , Mark D. Tully , José-maría Carazo , José L. Carrascosa , Pedro M. Matias , Tiago M. Bandeiras Scientific Reports 8 DOI: 10.1038/s41598-018-31997-z Diamond Proposal Number(s): [13193] Open Access Show Abstract ▼ Abstract: RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling.	Sep 2018
	Counterion binding alters surfactant self-assembly in deep eutectic solvents Adrian Sanchez-fernandez , O. S. Hammond , K. J. Edler , T. Arnold , J. Doutch , R. M. Dalgliesh , P. Li , K. Ma , A. J. Jackson Physical Chemistry Chemical Physics 114 DOI: 10.1039/C8CP01008K Show Abstract ▼ Abstract: Micellisation of surfactants in deep eutectic solvents has been recently demonstrated to provide a controllable way to modify micelle morphology. Ion-pair interactions between the solvent and the surfactant headgroup were identified as affecting the micellisation by modifying the charge density of the micelle. Here we explore the micellisation of dodecylsulfate surfactants with different counterions (Li+, Cs+, Mg2+, Bmim+, Emim+, cholinium+) dissolved in two deep eutectic solvents: choline chloride:urea and choline chloride:glycerol. Surface tension results show a solvent and counterion dependence of the CMC of the surfactants. Small-angle neutron scattering was subsequently used to investigate the morphology of the micelles formed. The results show that the elongation of the micelles is strongly dependent on the solvent, showing more elongated aggregates in choline chloride:urea than in choline chloride:glycerol. The morphology of micelles in DES was also found to depend on the counterion, where the affinity of binding showed similarities to that in water.	Apr 2018
B21-High Throughput SAXS	Multiple Conformational States Contribute to the 3D Structure of a Glucan Decasaccharide: A Combined SAXS and MD Simulation Study Sunhwan Jo , Daniel Myatt , Yifei Qi , James Doutch , Luke A. Clifton , Wonpil Im , Göran Widmalm The Journal Of Physical Chemistry B DOI: 10.1021/acs.jpcb.7b11085 Show Abstract ▼ Abstract: The inherent flexibility of carbohydrates is dependent on stereochemical arrangements, and characterization of their influence and importance will give insight into 3D structure and dynamics. In this study, a β-(1→4)/β-(1→3)-linked glucosyl decasaccharide is experimentally investigated by synchrotron small-angle X-ray scattering from which its radius of gyration (Rg) is obtained. Molecular dynamics (MD) simulations of the decasaccharide show four populated states at each glycosidic linkage, namely, syn- and anti-conformations. The calculated Rg values from the MD simulation reveal that in addition to syn-conformers, the presence of anti-ψ conformational states are required to reproduce experimental scattering data, unveiling inherent glycosidic linkage flexibility. The CHARMM36 force field for carbohydrates thus describes the conformational flexibility of the decasaccharide very well and captures the conceptual importance that anti-conformers are to be anticipated at glycosidic linkages of carbohydrates.	Dec 2017
	Surfactant-solvent interaction effects on the micellization of cationic surfactants in a carboxylic acid-based deep eutectic solvent Adrian Sanchez-fernandez , Oliver S. Hammond , Andrew J. Jackson , Thomas Arnold , James Doutch , Karen J. Edler Langmuir DOI: 10.1021/acs.langmuir.7b03254 Show Abstract ▼ Abstract: Deep eutectic solvents have been demonstrated to support amphiphile self-assembly, providing potential alternatives as structure directing agents in the synthesis of nanostructures, and drug delivery. Here we have expanded on this recent research to investigate the self-assembly of alkyltrimethylammonium bromide surfactants in choline chloride:malonic acid deep eutectic solvent and mixtures of the solvent with water. Surface tension and small-angle neutron scattering were used to determine the behavior of the amphiphiles. Surfactants were found to remain active in the solvent and surface tension measurements revealed changes in the behavior of the surfactants with different levels of hydration. Small-angle neutron scattering shows that in this solvent the micelle shape depends on the surfactant chain length, varying from globular micelles (aspect ratio ~2) for short chain surfactants to elongated micelles (aspect ratio ~14) for long chain surfactants even at low surfactant concentration. We suggest that the formation of elongated micelles can be explained through the interaction of the solvent with the surfactant headgroup, since ion-ion interactions between surfactant headgroups and solvent may modify the morphology of the micelles. The presence of water in the deep eutectic solvents promotes an increase in the charge density at the micelle interface and therefore the formation of less elongated, globular micelles.	Nov 2017
B21-High Throughput SAXS I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural Complexity in the KCTD Family of Cullin3-Dependent E3 Ubiquitin Ligases Daniel M. Pinkas , Caroline E. Sanvitale , Joshua C. Bufton , Fiona J. Sorrell , Nicolae Solcan , Rod Chalk , James Doutch , Alex N. Bullock Biochemical Journal DOI: 10.1042/BCJ20170527 Diamond Proposal Number(s): [8421] Show Abstract ▼ Abstract: Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5:5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.	Sep 2017
B21-High Throughput SAXS	Synthetic toxic Aß 1–42 oligomers can assemble in different morphologies Claude Bobo , Stéphane Chaignepain , Sarah Henry , Hélène Vignaud , Alfred Améadan , Christelle Marchal , Enora Prado , James Doutch , Jean-marie Schmitter , Corinne Nardin , Sophie Lecomte , Christophe Cullin Biochimica Et Biophysica Acta (bba) - General Subjects DOI: 10.1016/j.bbagen.2017.03.001 Diamond Proposal Number(s): [151] Show Abstract ▼ Abstract: Background Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aβ peptides. Aß toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aß1–42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. Methods Different biophysical methods (Fluorescence spectroscopy, Cross-linking, Mass spectrometry (MS), Small Angle X-Ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Calcein leakage) were used. Results The oligomers are mostly populated by a 14 mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aß amyloid fibrils. Conclusion The toxic synthetic AßG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel β-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aß dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes. General Significance The capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.	Mar 2017
B21-High Throughput SAXS	Mesoporous self-assembled nanoparticles of biotransesterified cyclodextrins and nonlamellar lipids as carriers of water-insoluble substances Leïla Zerkoune , Sylviane Lesieur , Jean-luc Putaux , Luc Choisnard , Annabelle Gèze , Denis Wouessidjewe , Borislav Angelov , Corinne Vebert-nardin , James Doutch , Angelina Angelova Soft Matter 12, 7539 - 7550 DOI: 10.1039/C6SM00661B Diamond Proposal Number(s): [10051] Open Access Show Abstract ▼ Abstract: Soft mesoporous hierarchically structured particles were created by the self-assembly of an amphiphilic deep cavitand cyclodextrin βCD-nC10 (degree of substitution n = 7.3), with a nanocavity grafted by multiple alkyl (C10) chains on the secondary face of the βCD macrocycle through enzymatic biotransesterification, and the nonlamellar lipid monoolein (MO). The effect of the non-ionic dispersing agent polysorbate 80 (P80) on the liquid crystalline organization of the nanocarriers and their stability was studied in the context of vesicle-to-cubosome transition. The coexistence of small vesicular and nanosponge membrane objects with bigger nanoparticles with inner multicompartment cubic lattice structures was established as a typical feature of the employed dispersion process. The cryogenic transmission electron microscopy (cryo-TEM) images and small-angle X-ray scattering (SAXS) structural analyses revealed the dependence of the internal organization of the self-assembled nanoparticles on the presence of embedded βCD-nC10 deep cavitands in the lipid bilayers. The obtained results indicated that the incorporated amphiphilic βCD-nC10 building blocks stabilize the cubic lattice packing in the lipid membrane particles, which displayed structural features beyond the traditional CD nanosponges. UV-Vis spectroscopy was employed to characterize the nanoencapsulation of a model hydrophobic dimethylphenylazo-naphthol guest compound (Oil red) in the created nanocarriers. In perspective, these dual porosity carriers should be suitable for co-encapsulation and sustained delivery of peptide, protein or siRNA biopharmaceuticals together with small molecular weight drug compounds or imaging agents.	Aug 2016
B21-High Throughput SAXS	Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level Daniel Coutandin , Christian Osterburg , Ratnesh Kumar Srivastav , Manuela Sumyk , Sebastian Kehrloesser , Jakob Gebel , Marcel Tuppi , Jens Hannewald , Birgit Schäfer , Eidarus Salah , Sebastian Mathea , Uta Müller-kuller , James Doutch , Manuel Grez , Stefan Knapp , Volker Dötsch Elife 5 DOI: 10.7554/eLife.13909 Diamond Proposal Number(s): [8421] Open Access Show Abstract ▼ Abstract: Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.	Mar 2016

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