BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments

Round, Adam; Felisaz, Franck; Fodinger, Lukas; Gobbo, Alexandre; Huet, Julien; Villard, C.; Blanchet, Clément E.; Pernot, Pétra; McSweeney, Seán; Roessle, Manfred; Svergun, Dmitri I.; Cipriani, F.

doi:10.1107/s1399004714026959

Cited by 193 publications

(151 citation statements)

References 14 publications

(15 reference statements)

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“…48,49 RSL bound to Mefuc or to Fuc-PEG was characterized at four different protein concentrations (10, 5, 2 and 1 mg/ml) using the automated sample changer. 50 Data were collected also on a sample of the conjugate that was pre-treated via an online SEC at BM29. 51 All of the samples were prepared in the same buffer, identical to that used for the NMR experiments.…”

Section: Saxs Characterizationmentioning

confidence: 99%

Noncovalent PEGylation via Lectin–Glycopolymer Interactions

et al. 2016

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The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 AbstractPEGylation, the covalent modification of proteins with polyethylene glycol, is an abundantly used technique to improve the pharmacokinetics of therapeutic proteins. The drawback with this methodology is that the covalently attached PEG can impede the biological activity (e.g. reduced receptor-binding capacity). Protein therapeutics with "disposable" PEG modifiers have potential advantages over the current technology. Here, we show that a protein-polymer "Medusa complex" is formed by the combination of a hexavalent lectin with a glycopolymer. Using NMR spectroscopy, small angle X-ray scattering (SAXS), size exclusion chromatography and native gel electrophoresis it was demonstrated that the fucose-binding lectin RSL and a fucose-capped polyethylene glycol (Fuc-PEG) form a multimeric assembly. All of the experimental methods provided evidence of noncovalent PEGylation with a concomitant increase in molecular mass and hydrodynamic radius. The affinity of the protein-polymer complex was determined by ITCand competition experiments to be in the µM range, suggesting that such systems have potential biomedical applications.

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Section: Saxs Characterizationmentioning

confidence: 99%

Noncovalent PEGylation via Lectin–Glycopolymer Interactions

et al. 2016

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“…Experiments were performed at European Synchrotron Radiation Facility (ESRF; Grenoble, France) Bio-SAXS beamline BM29 (16). To obtain the measurements, 30 l of protein solution at three different concentrations for each sample alternated with buffer measurements for background subtraction and the robotic sample handling available at the beamline were used (17). The experiments were programmed using the ISPyB BioSAXS interface (18), and the sequence was triggered via the BsxCuBE control software.…”

Section: Size Exclusion Chromatography (Sec) Combined With Detectionmentioning

confidence: 99%

Domain Organization of Vaccinia Virus Helicase-Primase D5

Hutin

Ling

Round

et al. 2016

J Virol

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Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single-and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design. V iruses of the family Poxviridae have a large linear doublestranded DNA (dsDNA) genome coding for up to 250 open reading frames. The double-stranded DNA genome is covalently circularized at both ends and is fully replicated in the cytoplasm. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) (1) and in the Phycodnaviridae (reviewed in reference 2). Like poxviruses, these viruses are also members of the nucleocytoplasmic large DNA virus (NCLDV) clade (3).We are working on vaccinia virus, which was used for the vaccination campaign that led to the eradication of smallpox. It is a convenient and rather safe system for the study of general poxviru...

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“…A series of lipoplexes with the LA12 surfactant concentration from 2 to 200 mM (p/n charge ratio: 1-100) was prepared. SAXS data (q-axis range 0.12 to 4.6 nm À1 , where q ¼ 4psinh/k, k ¼ 0.15 nm) were collected on the P12 beamline 18,19 at the Petra-III storage ring (DESYHamburg, Germany). The SAXS data were processed and analysed using PRIMUS program.…”

mentioning

confidence: 99%

The system with zwitterionic lactose-based surfactant for complexation and delivery of small interfering ribonucleic acid—A structural and spectroscopic study

Skupin

Sobczak

Zieliński

et al. 2016

Applied Physics Letters

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Systems suitable for the effective preparation of complexes with siRNA (small interfering RNA) are at the center of interest in the area of research work on the delivery of the RNA-based drugs (RNA-therapeutics). This article presents results of a study on the structural effects associated with siRNA complexation by a surfactant comprising a lactose group (N-(3-propanesulfone)-N-dodecyl-amino-beta-D-lactose hydrochloride, LA12). The double stranded siRNA oligomer (21 base pairs) used in this study is responsible for silencing a gene that can be important in the therapy of myotonic dystrophy type 1. The obtained siRNA/LA12 lipoplexes were studied using the methods of small angle scattering of synchrotron radiation, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and electrophoretic mobility tests. Lipoplexes form in solution stable lamellar or cubic phases. The surfactant selected for the study shows much lower cytotoxicity and good complexation abilities of siRNA than dicationic or polycationic surfactants.

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BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments

Cited by 193 publications

References 14 publications

Noncovalent PEGylation via Lectin–Glycopolymer Interactions

Noncovalent PEGylation via Lectin–Glycopolymer Interactions

Domain Organization of Vaccinia Virus Helicase-Primase D5

The system with zwitterionic lactose-based surfactant for complexation and delivery of small interfering ribonucleic acid—A structural and spectroscopic study

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