Protein aggregation is of broad importance in biotechnology and disease, yet the structural heterogeneity of cellular aggregates has confounded high-resolution structural analysis. Inclusion bodies (IBs) formed in Escherichia coli are an attractive, controllable system for unravelling the complexities of protein aggregation in a cellular context. Here, a multimodal analysis integrating residue-resolved quenched amide hydrogen-deuterium exchange (qHDX), proteolysis, FTIR, Congo red binding, and chemical denaturation is applied to IBs formed by proteins encompassing stable beta- and alpha- globular folds, a partially structured protein fragment, and intrinsically disordered low complexity domains (LCDs). Remarkable conformational diversity is observed: IBs formed by well-folded proteins are extensively structured and include substantial local native-like features, whereas proteins with decreased access to stable native conformations form more heterogeneous and dynamic aggregates increasingly shaped by intrinsic sequence features. Strikingly, qHDX protection of TDP-43 LCD IBs strongly aligns with the core of cryo-EM structures of ex vivo pathological fibrils; however, peripheral regions that appear fully hydrogen-bonded in the cryo-EM structures exhibit little protection. The results reveal that individual protein IBs contain distinct mixtures of native-like, disordered, and amyloid-like conformers, informing prediction and control of cellular aggregate structure and stability.
Siebeneichler, B., Liu, X., Rodriguez Cruz, P. E., Naser, D., DelMistro, G., Steckner, J., Schaefer, A., Tran, N., Holyoak, T., Meiering, E. M.
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