Protein cages are supramolecular assemblies found across all domains of life, where they play essential biological roles such as iron storage, genome encapsulation and protection, and enzyme compartmentalization. Their well-defined architecture, biocompatibility, and amenability to modification have made them attractive scaffolds for applications in drug delivery, imaging, and nanomaterials. Ferritin is a ubiquitous protein cage that naturally functions to store iron and limit its redox toxicity by oxidizing Fe²⁺ and depositing the iron oxide mineral within an internal cavity. While prior studies have elucidated iron transport pathways, iron oxidation steps, and iron mineral nucleation events, management of the proton “byproducts” of iron biomineralization remains poorly understood.This dissertation presents two complementary investigations into the functional versatility of archaeal ferritin from Archaeoglobus fulgidus (AfFtn), which has unique ionic strength-dependent assembly properties. Part one focuses on spatiotemporal proton dynamics during iron biomineralization. We site-specifically introduced interior-surface cysteines near the ferroxidase centers and labeled them with fluorescein. These internal sensors, along with an external fluorescein-labeled peptide, enabled real-time tracking of apparent pH changes. Our results revealed that most protons generated during Fe²⁺ oxidation are released to bulk solution on both short (<10 s) and longer (minutes) timescales, with only a small fraction retained within the ferritin cavity. These findings suggest that proton retention is a previously underappreciated component of ferritin’s biological function, potentially influencing iron mineralization and the ferritin reactor microenvironment. Part two investigates how the electrostatic properties of protein cargo influence ferritin assembly. Guided by molecular dynamics (MD) simulations and computational protein design, we found that both positive charge magnitude and distribution critically govern the propensity for and efficiency of cargo-induced assembly. Our data provided the first example of cooperative assembly with a ferritin capsule. Furthermore, we established a strategy to construct stoichiometric 1:1 protein host–guest complexes, confirmed by time-resolved fluorescence anisotropy. Our approach provides a platform for generating homogeneous host–guest complexes with tunable properties for future applications. Together, these studies expand our understanding of ferritin function and establish new principles for engineering protein cage assemblies through control of host–guest interactions.