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Article ; Online: Using atomistic solution scattering modelling to elucidate the role of the Fc glycans in human IgG4.

Spiteri, Valentina A / Doutch, James / Rambo, Robert P / Bhatt, Jayesh S / Gor, Jayesh / Dalby, Paul A / Perkins, Stephen J

PloS one

2024 Volume 19, Issue 4, Page(s) e0300964

Abstract: ... after deglycosylation were almost unchanged. In the P(r) distance distribution curves, the two M1 and M2 ...

Abstract	Human immunoglobulin G (IgG) exists as four subclasses IgG1-4, each of which has two Fab subunits joined by two hinges to a Fc subunit. IgG4 has the shortest hinge with 12 residues. The Fc subunit has two glycan chains, but the importance of glycosylation is not fully understood in IgG4. Here, to evaluate the stability and structure of non-glycosylated IgG4, we performed a multidisciplinary structural study of glycosylated and deglycosylated human IgG4 A33 for comparison with our similar study of human IgG1 A33. After deglycosylation, IgG4 was found to be monomeric by analytical ultracentrifugation; its sedimentation coefficient of 6.52 S was reduced by 0.27 S in reflection of its lower mass. X-ray and neutron solution scattering showed that the overall Guinier radius of gyration RG and its cross-sectional values after deglycosylation were almost unchanged. In the P(r) distance distribution curves, the two M1 and M2 peaks that monitor the two most common distances within IgG4 were unchanged following deglycosylation. Further insight from Monte Carlo simulations for glycosylated and deglycosylated IgG4 came from 111,382 and 117,135 possible structures respectively. Their comparison to the X-ray and neutron scattering curves identified several hundred best-fit models for both forms of IgG4. Principal component analyses showed that glycosylated and deglycosylated IgG4 exhibited different conformations from each other. Within the constraint of unchanged RG and M1-M2 values, the glycosylated IgG4 models showed more restricted Fc conformations compared to deglycosylated IgG4, but no other changes. Kratky plots supported this interpretation of greater disorder upon deglycosylation, also observed in IgG1. Overall, these more variable Fc conformations may demonstrate a generalisable impact of deglycosylation on Fc structures, but with no large conformational changes in IgG4 unlike those seen in IgG1.
MeSH term(s)	Humans ; Immunoglobulin G/chemistry ; Cross-Sectional Studies ; Models, Molecular ; Immunoglobulin Fc Fragments/chemistry
Chemical Substances	Immunoglobulin G ; Immunoglobulin Fc Fragments
Language	English
Publishing date	2024-04-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0300964
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Considerations for Sample Preparation Using Size-Exclusion Chromatography for Home and Synchrotron Sources.

Rambo, Robert P

Advances in experimental medicine and biology

2015 Volume 1009, Page(s) 31–45

Abstract: The success of a SAXS experiment for structural investigations depends on two precise measurements, the sample and the buffer background. Buffer matching between the sample and background can be achieved using dialysis methods but in biological SAXS of ... ...

Abstract	The success of a SAXS experiment for structural investigations depends on two precise measurements, the sample and the buffer background. Buffer matching between the sample and background can be achieved using dialysis methods but in biological SAXS of monodisperse systems, sample preparation is routinely being performed with size exclusion chromatography (SEC). SEC is the most reliable method for SAXS sample preparation as the method not only purifies the sample for SAXS but also almost guarantees ideal buffer matching. Here, I will highlight the use of SEC for SAXS sample preparation and demonstrate using example proteins that SEC purification does not always provide for ideal samples. Scrutiny of the SEC elution peak using quasi-elastic and multi-angle light scattering techniques can reveal hidden features (heterogeneity) of the sample that should be considered during SAXS data analysis. In some cases, sample heterogeneity can be controlled using a small molecule additive and I outline a simple additive screening method for sample preparation.
MeSH term(s)	Buffers ; Chromatography, Gel/instrumentation ; Chromatography, Gel/methods ; Excipients/chemistry ; Humans ; Phosphates/chemistry ; Protein Aggregates ; Protein Conformation ; Proteins/chemistry ; Proteins/ultrastructure ; Scattering, Small Angle ; Specimen Handling/methods ; Sucrose/chemistry ; Synchrotrons/instrumentation ; X-Ray Diffraction/instrumentation ; X-Ray Diffraction/methods ; X-Ray Diffraction/standards
Chemical Substances	Buffers ; Excipients ; Phosphates ; Protein Aggregates ; Proteins ; Sucrose (57-50-1)
Language	English
Publishing date	2015-05-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2214-8019 ; 0065-2598
ISSN (online)	2214-8019
ISSN	0065-2598
DOI	10.1007/978-981-10-6038-0_3
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Resolving Individual Components in Protein-RNA Complexes Using Small-Angle X-ray Scattering Experiments.

Rambo, Robert P

Methods in enzymology

2015 Volume 558, Page(s) 363–390

Abstract: Small-angle X-ray scattering (SAXS) of protein-RNA complexes has developed into an efficient and economical approach for determining low-resolution shapes of particles in solution. Here, we demonstrate a mutliphase volumetric modeling approach capable of ...

Abstract	Small-angle X-ray scattering (SAXS) of protein-RNA complexes has developed into an efficient and economical approach for determining low-resolution shapes of particles in solution. Here, we demonstrate a mutliphase volumetric modeling approach capable of resolving individual components within a low-resolution shape. Through three case studies, we describe the SAXS data collecting strategies, premodeling analysis, and computational methods required for deconstructing complexes into their respective components. This chapter presents an approach using the programs ScÅtter and MONSA and custom scripts for averaging and aligning of multiple independent modeling runs. The method can image small (7kDa) masses within the context of complex and is capable of visualizing ligand-induced conformational changes. Nevertheless, computational algorithms are not without error, and we describe specific considerations during SAXS data reduction and modeling to mitigate possible false positives.
MeSH term(s)	Algorithms ; Binding Sites ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA Helicases/chemistry ; DNA Helicases/genetics ; DNA Helicases/metabolism ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Humans ; Ligands ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; Protein Binding ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA Precursors/chemistry ; RNA Precursors/genetics ; RNA Precursors/metabolism ; RNA Splicing ; RNA Splicing Factors ; RNA, Transfer/chemistry ; RNA, Transfer/genetics ; RNA, Transfer/metabolism ; Scattering, Small Angle ; Software ; Thermodynamics ; Transcription Factors/chemistry ; Transcription Factors/genetics ; Transcription Factors/metabolism ; X-Ray Diffraction ; X-Rays
Chemical Substances	DNA-Binding Proteins ; Ligands ; RNA Precursors ; RNA Splicing Factors ; SF1 protein, human ; Transcription Factors ; DNA (9007-49-2) ; RNA, Transfer (9014-25-9) ; SMARCAL1 protein, human (EC 2.7.7.-) ; DNA Helicases (EC 3.6.4.-)
Language	English
Publishing date	2015-04-03
Publishing country	United States
Document type	Journal Article
ISSN	1557-7988 ; 0076-6879
ISSN (online)	1557-7988
ISSN	0076-6879
DOI	10.1016/bs.mie.2015.02.004
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Analysis of SEC-SAXS data via EFA deconvolution and Scatter.

Tully, Mark D / Tarbouriech, Nicolas / Rambo, Robert P / Hutin, Stephanie

Journal of visualized experiments : JoVE

2021 , Issue 167

Abstract: BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS ... ...

Abstract	BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.
MeSH term(s)	Algorithms ; Chromatography, Gel ; DNA/chemistry ; Data Analysis ; Proteins/chemistry ; Scattering, Small Angle ; X-Ray Diffraction
Chemical Substances	Proteins ; DNA (9007-49-2)
Language	English
Publishing date	2021-01-28
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/61578
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Article ; Online: Biophysical characterization of the structure of a SARS-CoV-2 self-amplifying RNA (saRNA) vaccine.

Myatt, Daniel P / Wharram, Lewis / Graham, Charlotte / Liddell, John / Branton, Harvey / Pizzey, Claire / Cowieson, Nathan / Rambo, Robert / Shattock, Robin J

Biology methods & protocols

2023 Volume 8, Issue 1, Page(s) bpad001

Abstract: The current SARS-Covid-2 (SARS-CoV-2) pandemic has led to an acceleration of messenger ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA), has ... ...

Abstract	The current SARS-Covid-2 (SARS-CoV-2) pandemic has led to an acceleration of messenger ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA), has required concomitant development of analytical characterization techniques. Characterizing the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This article describes the biophysical characterization of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterize the IMP-1 RNA molecule. In this article, we use ultraviolet spectroscopy, dynamic light scattering, size-exclusion chromatography small-angle X-ray scattering and circular dichroism to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule.
Language	English
Publishing date	2023-02-14
Publishing country	England
Document type	Journal Article
ISSN	2396-8923
ISSN (online)	2396-8923
DOI	10.1093/biomethods/bpad001
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Solution structure of deglycosylated human IgG1 shows the role of C

Spiteri, Valentina A / Doutch, James / Rambo, Robert P / Gor, Jayesh / Dalby, Paul A / Perkins, Stephen J

Biophysical journal

2021 Volume 120, Issue 9, Page(s) 1814–1834

Abstract: The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are ... ...

Abstract	The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s
MeSH term(s)	Cross-Sectional Studies ; Humans ; Immunoglobulin G ; Models, Molecular ; Polysaccharides ; Protein Conformation ; Receptors, IgG
Chemical Substances	Immunoglobulin G ; Polysaccharides ; Receptors, IgG
Language	English
Publishing date	2021-03-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2021.02.038
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Article ; Online: Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses.

Spiteri, Valentina A / Goodall, Margaret / Doutch, James / Rambo, Robert P / Gor, Jayesh / Perkins, Stephen J

The Journal of biological chemistry

2021 Volume 297, Issue 3, Page(s) 100995

Abstract: Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role ... ...

Abstract	Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s
MeSH term(s)	Amino Acid Sequence ; Chromatography, Liquid/methods ; Glycosylation ; Humans ; Immunoglobulin Fab Fragments/chemistry ; Immunoglobulin G/chemistry ; Mass Spectrometry/methods ; Molecular Dynamics Simulation ; Multiple Myeloma/immunology ; Myeloma Proteins/chemistry ; Neutrons ; Protein Conformation ; Receptors, Fc/chemistry ; Scattering, Small Angle ; Sequence Homology, Amino Acid ; Ultracentrifugation/methods ; X-Ray Diffraction
Chemical Substances	Immunoglobulin Fab Fragments ; Immunoglobulin G ; Myeloma Proteins ; Receptors, Fc ; myeloma immunoglobulins
Language	English
Publishing date	2021-07-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2021.100995
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Article ; Online: The SKMT Algorithm

Arron Bale / Robert Rambo / Christopher Prior

PLoS Computational Biology, Vol 19, Iss 11, p e

A method for assessing and comparing underlying protein entanglement.

2023 Volume 1011248

Abstract: We present fast and simple-to-implement measures of the entanglement of protein tertiary structures which are appropriate for highly flexible structure comparison. These are performed using the SKMT algorithm, a novel method of smoothing the Cα backbone ... ...

Abstract	We present fast and simple-to-implement measures of the entanglement of protein tertiary structures which are appropriate for highly flexible structure comparison. These are performed using the SKMT algorithm, a novel method of smoothing the Cα backbone to achieve a minimal complexity curve representation of the manner in which the protein's secondary structure elements fold to form its tertiary structure. Its subsequent complexity is characterised using measures based on the writhe and crossing number quantities heavily utilised in DNA topology studies, and which have shown promising results when applied to proteins recently. The SKMT smoothing is used to derive empirical bounds on a protein's entanglement relative to its number of secondary structure elements. We show that large scale helical geometries dominantly account for the maximum growth in entanglement of protein monomers, and further that this large scale helical geometry is present in a large array of proteins, consistent across a number of different protein structure types and sequences. We also show how these bounds can be used to constrain the search space of protein structure prediction from small angle x-ray scattering experiments, a method highly suited to determining the likely structure of proteins in solution where crystal structure or machine learning based predictions often fail to match experimental data. Finally we develop a structural comparison metric based on the SKMT smoothing which is used in one specific case to demonstrate significant structural similarity between Rossmann fold and TIM Barrel proteins, a link which is potentially significant as attempts to engineer the latter have in the past produced the former. We provide the SWRITHE interactive python notebook to calculate these metrics.
Keywords	Biology (General) ; QH301-705.5
Subject code	612
Language	English
Publishing date	2023-11-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Modeling macromolecular motions by x-ray-scattering-constrained molecular dynamics.

Rambo, Robert P / Tainer, John A

Biophysical journal

2015 Volume 108, Issue 10, Page(s) 2421–2423

MeSH term(s)	Aspartate Carbamoyltransferase/chemistry ; Karyopherins/chemistry ; Molecular Dynamics Simulation ; Scattering, Small Angle ; X-Ray Diffraction/methods
Chemical Substances	Karyopherins ; Aspartate Carbamoyltransferase (EC 2.1.3.2)
Language	English
Publishing date	2015-05-19
Publishing country	United States
Document type	Comment ; Journal Article
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2015.04.023
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Article: Analysis of sec-saxs data via efa deconvolution and scatter

Tully, Mark D / Tarbouriech, Nicolas / Rambo, Robert P / Hutin, Stephanie

Journal of visualized experiments. 2021 Jan. 28, , no. 167

2021

Abstract	BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.
Keywords	DNA-directed DNA polymerase ; data collection ; gel chromatography ; mutants ; particle size ; structural biology
Language	English
Dates of publication	2021-0128
Size	p. e61578.
Publishing place	Journal of Visualized Experiments
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/61578
Database	NAL-Catalogue (AGRICOLA)

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