Quenched hydrogen-deuterium amide exchange optimization for high-resolution structural analysis of cellular protein aggregates.

Inclusion bodies (IBs) are large, insoluble aggregates that often form during the overexpression of proteins in bacteria. These aggregates are of broad fundamental and practical significance, for recombinant protein preparation and due to their relevance to aggregation-related medical conditions and their recent emergence as promising functional nanomaterials. Despite their significance, high resolution knowledge of IB structure remains very limited. Such knowledge will advance understanding and control of IB formation and properties in myriad practical applications. Here, we report a detailed quenched hydrogen-deuterium amide exchange (qHDX) method with NMR readout to define the structure of IBs at the level of individual residues throughout the protein. Applying proper control of experimental conditions, such as sample pH, water content, temperature, and intrinsic rate of amide exchange, yields in depth results for these cellular protein aggregates. qHDX results illustrated for Cu, Zn superoxide dismutase 1 (SOD1) and Adnectins show their IBs include native-like structure and some but not all mutations alter IB structure.