scholarly journals Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 48
Author(s):  
Frank Lüdel ◽  
Sandra Bufe ◽  
Willem M. Bleymüller ◽  
Hugo de de Jonge ◽  
Luisa Iamele ◽  
...  
Keyword(s):  
Small Angle ◽  
Antibody Fragment ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Single Chain ◽  
X Ray ◽  
X Ray Scattering ◽  
Single Chain Fv ◽  
Exclusion Chromatography ◽  
Ray Scattering

Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

scholarly journals Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

2018 ◽  
Vol 51 (6) ◽  
pp. 1623-1632 ◽  
Author(s):  
Saskia Bucciarelli ◽  
Søren Roi Midtgaard ◽  
Martin Nors Pedersen ◽  
Søren Skou ◽  
Lise Arleth ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering Data ◽  
X Ray ◽  
Laboratory Instrument ◽  
X Ray Scattering ◽  
Home Institution ◽  
Exclusion Chromatography ◽  
Ray Scattering

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

scholarly journals Newly developed Laboratory-based Size exclusion chromatography Small-angle x-ray scattering System (La-SSS)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rintaro Inoue ◽  
Tatsuo Nakagawa ◽  
Ken Morishima ◽  
Nobuhiro Sato ◽  
Aya Okuda ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering System ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Ray Scattering

scholarly journals Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

2015 ◽  
Vol 11 (1) ◽  
pp. 190-196 ◽  
Author(s):  
Cyril C. Curtain ◽  
Nigel M. Kirby ◽  
Haydyn D. T. Mertens ◽  
Kevin J. Barnham ◽  
Robert B. Knott ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Alpha Synuclein ◽  
Size Exclusion ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Modelling Study ◽  
Ray Scattering

Size exclusion chromatography with small angle X-ray scattering and ensemble optimisation modelling reveals conformers in random pool of α-synuclein.

scholarly journals Integrated beamline control and data acquisition for small-angle X-ray scattering at the P12 BioSAXS beamline at PETRAIII storage ring DESY

2018 ◽  
Vol 25 (3) ◽  
pp. 906-914 ◽  
Author(s):  
Nelly R. Hajizadeh ◽  
Daniel Franke ◽  
Dmitri I. Svergun
Keyword(s):  
Data Acquisition ◽  
Small Angle ◽  
Size Exclusion ◽  
Full Potential ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Novice Users ◽  
Intuitive Interface ◽  
Ray Scattering

The versatility of small-angle X-ray scattering (SAXS) as a structural biology method is apparent by its compatibility with many experimental set-ups. Most advanced SAXS studies are conducted at dedicated synchrotron beamlines yielding high beam brilliance, throughput and temporal resolution. However, utilizing the full potential of the method while preserving a high degree of automation provides a challenge to any SAXS beamline. This challenge is especially pertinent at the P12 BioSAXS beamline of the EMBL at the PETRAIII Synchrotron DESY (Hamburg, Germany), optimized and dedicated to scattering of macromolecular solutions. Over 200 unique set-ups are possible at this beamline offering various functionalities, including different temporal and spatial resolutions. Presented here is a beamline control and data-acquisition software,BECQUEREL, designed to maximize flexibility and automation in the operation of P12. In the frame of a single intuitive interface the control system allows for convenient operation with all hardware set-ups available at P12 including a robotic sample changer, in-line size-exclusion chromatography, stop-flow devices, microfluidic spinning disk and various in-air settings. Additional functionalities are available to assist the data-collection procedure for novice users, and also routine operation of the support staff.

scholarly journals Methods for analysis of size-exclusion chromatography–small-angle X-ray scattering and reconstruction of protein scattering

2015 ◽  
Vol 48 (4) ◽  
pp. 1102-1113 ◽  
Author(s):  
Andrew W. Malaby ◽  
Srinivas Chakravarthy ◽  
Thomas C. Irving ◽  
Sagar V. Kathuria ◽  
Osman Bilsel ◽  
...  
Keyword(s):  
Linear Combination ◽  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Data Sets ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Ray Scattering

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC–SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC–SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC–SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD–LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known asData Evaluation and Likelihood Analysis(DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.

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