Surface functionalization with short PAS-sequence affects H-ferritin nanocage stability

H-ferritin nanocages (HFn) are promising tumor-targeting delivery systems owing to their specificity and reduced off-target toxicity. However, their short half-life and rapid clearance currently limit clinical application. PASylation has been used to increase the in vivo half-life of HFn, without introducing negative effects. To date, all knowledge is limited to PASylated HFn containing PAS domains of 40 and 75 amino acids. Herein, we investigate the impact of shorter PAS domains on the stability of HFn, focusing on a mutant modified with 20 amino acids PAS domains. PAS20-HFn monomers were successfully produced and purified but seems unable to form stable quaternary structure. To investigate this, we studied the structural dynamics properties of native HFn, PAS20-HFn, PAS40-HFn dimers, tetramers, and octamers. Our simulations indicate that PAS20-HFn tetramers exhibit increased flexibility in a region encompassing the end of the D helix, the D-E turn and the beginning of the E helix. This flexibility is critical for dimer interactions and facilitates the subsequent organization of higher-order complexes. During the folding of PAS20-HFn octamers, PAS domain disrupts the proper association of the Cterminal E-helices, leading to instability in the nanocages. This highlights the bipolar and essential role of HFn PASylation in HFn nanocages, especially concerning their size.