Charge engineering controls cooperative assembly and loading in protein host–guest complexes

Controlling cargo loading in self-assembling protein capsules remains a key challenge in supramolecular chemistry. Inspired by nature's capacity for host-guest recognition, we engineered supercharged green fluorescent protein (GFP) cargo for controlling its encapsulation by <i>Archaeoglobus fulgidus</i> ferritin. Guided by molecular dynamics (MD) simulations and computational protein design, experiments confirmed that GFP charge magnitude and distribution dictate capsule assembly and loading efficiency. These data provide the first example of cooperative assembly with a ferritin capsule. Finally, we established a strategy for generating stoichiometric 1 : 1 protein host-guest complexes, confirmed by time-resolved fluorescence anisotropy. This provides a blueprint for designing ferritin host-guest complexes with enhanced homogeneity and functionality.