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An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism

Codina, N; Hilton, D; Zhang, C; Chakroun, N; Ahmad, SS; Perkins, SJ; Dalby, PA; (2019) An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism. Journal of Molecular Biology 10.1016/j.jmb.2019.02.009. (In press). Green open access

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Abstract

Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states, is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states - notably amyloids - have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle X-ray scattering (SAXS), atomistic molecular dynamics (MD) simulations, single-molecule FRET (smFRET), and aggregation-prone region (APR) predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, Rg, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full molecular dynamics simulations from the same conditions, and located the conformational changes in the native state to the constant domain of the light chain (CL). This CL displacement was independently confirmed using smFRET measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted aggregation-prone region (APR), suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation.

Type: Article
Title: An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.jmb.2019.02.009
Publisher version: https://doi.org/10.1016/j.jmb.2019.02.009
Language: English
Additional information: © 2019 The Author(s). Published by Elsevier Ltd. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed.
Keywords: Antibody fragment, Molecular dynamics, Protein aggregation, Single-molecule FRET, X-ray scattering
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Chemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10068999
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