Peptide‐Ligand Cooperative Interplay Drives Gold Nanoparticle Encapsulation by Protein Cages

ABSTRACT Cargo encapsulation offers broad opportunities in synthetic biology, biocatalysis, and therapeutic delivery, with encapsulins serving as nanoscale reaction chambers or protective carriers. Yet, controlling cargo loading remains challenging. Here, we reveal a molecular‐scale understanding of gold nanoparticle encapsulation in encapsulin protein cages. Experiments investigate how salt concentration, nanoparticle functionalization with ligands, and cargo‐loading peptides influence encapsulation performance. Molecular dynamics simulations connect these experimental observations to the free‐energy landscape governing the initial association of an encapsulin protomer binding to the nanoparticle surface. Simulations reveal three salt‐dependent sets of nanoparticle‐protomer binding free‐energy compared to protomer‐protomer binding energy: much stronger nanoparticle–protomer binding at low salt, slightly stronger nanoparticle–protomer binding in a wide range of intermediate salt, and weakened nanoparticle–protomer attraction at high salt, corresponding to experimental observations of co‐precipitates, nanoparticle encapsulation, and empty cages, respectively. Importantly, the robustness of encapsulation to variations in salt concentration arises from cooperative effects between ligands and peptides: ligands mediate electrostatic attraction and promote peptide extension, while peptides extend the protomer recruitment zone and prevent kinetic trapping. This integrated experimental and computational approach provides molecular‐level insight into encapsulation energetics and peptide‐ligand cooperative interplay, guiding the rational design of bio‐inspired nanocages for selective delivery and templated synthesis.