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

• DOI: 10.1016/j.jmb.2019.02.009
• Publication Date: 2019-02-15T21:41:44Z
• Authors: Nuria Codina; David Hilton; Cheng Zhang; Nesrine Chakroun; Shahina S. Ahmad; Stephen J. Perkins; Paul A. Dalby
• Keywords: Small-angle X-ray scattering; Radius of gyration; Conformational ensembles; Energy landscape; Single-molecule FRET

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