Divergent Inclusion Body Structures and Stabilities Emerge from Native Monomer Properties

Siebeneichler, B.; Liu, X.; Rodriguez Cruz, P. E.; Naser, D.; DelMistro, G.; Steckner, J.; Schaefer, A.; Tran, N.; Holyoak, T.; Meiering, E. M.

doi:10.64898/2026.06.01.729379

bioRxiv · 10.64898/2026.06.01.729379

Divergent Inclusion Body Structures and Stabilities Emerge from Native Monomer Properties

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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Abstract

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 -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 the prediction and control of cellular aggregate structure and stability. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/729379v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1542626org.highwire.dtl.DTLVardef@194ad5forg.highwire.dtl.DTLVardef@26c13org.highwire.dtl.DTLVardef@1055573_HPS_FORMAT_FIGEXP M_FIG Inclusion body conformational heterogeneity. Left: aggregates retaining local native-like structure. Middle: assemblies populated by -helical intermediates coexisting with antiparallel -sheet oligomers. Right: initially disordered states undergo helix-helix interactions and progress towards -sheet-rich conformations, illustrating distinct routes of cellular aggregation. C_FIG

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Siebeneichler, B., Liu, X., Rodriguez Cruz, P. E., Naser, D., DelMistro, G., Steckner, J., Schaefer, A., Tran, N., Holyoak, T., Meiering, E. M.. 2026-06-02. Divergent Inclusion Body Structures and Stabilities Emerge from Native Monomer Properties. https://doi.org/10.64898/2026.06.01.729379

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