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 dynamic simulations, single-molecule Förster resonance energy transfer, and aggregation-prone region 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 MD 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 single-molecule Förster resonance energy transfer measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted 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.
Keywords: antibody fragment; molecular dynamics; protein aggregation; single-molecule FRET; x-ray scattering.
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