Rational Design of Stable, Dual-Functionalized Ferritin Nanocages via Molecular Dynamics-Guided In Vitro Heteromeric Assembly

Abstract This study presents a rational, simulation-guided strategy to overcome the structural instability of ferritin nanocages upon conjugation with large biomoleculesa common challenge in protein-based bioconjugates. Molecular dynamics (MD) simulations were performed using epidermal growth factor (EGF) and enhanced green fluorescent protein (eGFP) as models, which predicted that excessive eGFP fusion induces steric hindrance, disrupts hydrogen bonding, and weakens binding free energy at critical C3 interfaces, thereby destabilizing the cage. Based on these insights, an in vitro heteromeric assembly strategy was designed, employing EGF- and eGFP-(G4S)3-FTH1 subunits to generate pure, dual-functional nanostructures as a genetically programmable alternative to chemical bioconjugation. Successful assembly and functionality were confirmed by electron microscopy, fluorescence, and cell binding assays. To quantitatively assess stability, comprehensive biophysical profiling was performed under thermal, pH, and salt stress. Thermal denaturation, as monitored by nanoDSF, native PAGE, and qPCR-DSF, revealed a stepwise disassembly pathway: early local rearrangements at C3 interfaces precede global cage dissociation, and the disassembly midpoint temperature (Tdiss) of the intact 24-mer increased from 62.9 °C to 79.2 °C as the EGF-FTH1 proportion increased, consistent with MD-predicted weakening of C3-interface hydrogen bonds and binding energy. Although a proportion of 40% EGF-FTH1 subunits (with 60% eGFP-(G4S)3-FTH1) represented the minimal threshold for forming intact heteromeric cages, 60% EGF-FTH1 (with 40% eGFP-(G4S)3-FTH1) provided the optimal balance between functional display and stability, retaining >80% cage integrity at pH 7, exhibiting superior salt tolerance, and maintaining one-month storage stability at 4 °C; increasing the eGFP-(G4S)3-FTH1 ratio beyond 40% led to markedly reduced stability. The co-assembly approach was validated with TAT/EGF-functionalized cages, which maintained stability and enhanced cellular uptake. MD-predicted subunit interfaces were thus used to guide the combination of stable and unstable subunit pairs, yielding protein nanocages with balanced functionality and stability, and establishing this strategy as a versatile approach for expanding the utility of multifunctional protein nanocages in biomedicine and industrial biocatalysis.