Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

Blanchet, Clément E.; Spilotros, Alessandro; Schwemmer, F.; Graewert, Melissa A.; Kikhney, Alexey; Jeffries, Cy M.; Franke, Daniel; Mark, Daniel; Zengerle, Roland; Cipriani, F.; Fiedler, S.; Roessle, Manfred; Svergun, Dmitri I.

doi:10.1107/s160057671500254x

Cited by 548 publications

(454 citation statements)

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“…34 Using a PILATUS 2M pixel detector at a sampledetector distance of 3.1 m and at an energy of 10 keV (λD 1.24 A ), the range of momentum transfer 0.01 < s < 0.45 A ¡1 was covered (s D 4p sinu/λ, where 2u is the scattering angle). The initial stock solutions in D-PBS (Gibco Life Technologies) were measured at 24 mg/ml and 28 mg/ml, respectively, as well as concentration series hereof.…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Plath

Ringler

Graff-Meyer

et al. 2016

mAbs

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(2016) Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs, mAbs, 8:5, 928-940, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTThe formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

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Section: Transmission Electron Microscopymentioning

confidence: 99%

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Plath

Ringler

Graff-Meyer

et al. 2016

mAbs

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“…SAXS requires small (milligram) amounts of purified and monodisperse samples. The experiment can be performed rapidly using the dedicated beamlines at synchrotron light sources [2]. Even data analysis, when the quality of the sample is good enough, can be fast, thanks to powerful specialized software [3].…”

Section: Introductionmentioning

confidence: 99%

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

et al. 2018

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Abstract:The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N-and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

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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.…”

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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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Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

Cited by 548 publications

References 50 publications

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

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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