Ferritin-based hybrid macromolecules experience unusual shift of stoichiometry distribution

Ferritin-based hybrids are large 24-subunit macromolecules of megadalton scale have prospective applications ranging from drug delivery to recombinant vaccines, however, their rational design is challenging. Here, we architectured hybrids based on ferritin subunits from Helicobacter pylori and ones fused with a homolog of the Small Ubiquitin-like Modifier protein. We firstly revealed the stochastic nature of bacterial ferritin-based hybrids self-assembly by observing a sequential range of stoichiometries at totally different sample preparation procedures: coexpression in Escherichia coli cells and pH-dependent dis/reassembly. We developed an approach of quantitative evaluation of stoichiometry distribution by using a model based on random, unambiguous, and stoichiometry-independent assembly of hexamers into 24-meric hybrid globules. We identified the presence of a heterodimer and found unexpectedly disfavored stoichiometries of hexamers, which determined the narrowing of structural diversity patterns of 24-mers and shifted the stoichiometry distribution from the random one. Our findings provide new insights into the molecular mechanisms governing the shift in structural diversity patterns of ferritin-based hybrid globules. Finally, the combination of our model system and the hexamer-based approach provides a robust platform for the rational design of ferritin-based systems, with potential applications in drug delivery, structure-based immunogen design, and beyond.