Electrostatic Patterning Controls Mineral Nucleation Inside Ferritin

Ferritin protein nanocages store iron across nearly all living organisms. In mammals, two subtypes of ferritin exist: heavy (H) chain and light (L) chain. They have very similar 3D structures, but each performs a slightly different role in iron mineral formation. How sequence differences between the two subtypes affect mineral formation within the nanocages is still unclear. Single-particle reconstruction of cryo-TEM images was used to build models of unmineralized and partially mineralized human L-chain and H-chain ferritin, which showed that subtle differences in protein structure led to changes in the location of mineral formation within ferritin. Explicit-solvent atomistic molecular dynamics (MD) simulations were used to explore how sequence-dependent electrostatics modulate ion transport, cluster formation, and mineral nucleation within the confined environment of human L- and H-chain apoferritin nanocages. Employing NaCl as a computational probe, we show that the internal charge distribution governs ion selectivity and nucleation pathways. Analysis of liquid and solid ionic clusters, combined with Markov State Models (MSMs), reveals that mineralization proceeds through a two-step mechanism involving dense liquid-like precursors that crystallize homogeneously within the cavity. These findings provide molecular insight into how ferritin sequence variability tunes confinement-driven nucleation and suggest general principles for designing biomimetic nanoreactors.