Tryptophan Residues' Incorporation Modulates Ferritin Thermal Stability and Hydrophobicity

Ferritin, a physiological iron-storage protein, has emerged as a highly attractive platform for drug delivery owing to its biocompatibility, structural robustness, and intrinsic ability to encapsulate and protect therapeutic cargo within its hollow nanocage. Building upon previous studies that established the baseline characteristics of engineered ferritin mutants in comparison to the wild-type protein, this work specifically investigates and directly compares the thermal stability profiles of two distinct mutated variants. These variants of human H-chain ferritin, obtained through targeted site-directed mutagenesis, feature either four or six tryptophan residues per subunit, strategically positioned toward the inner cavity of the protein shell. These modifications were intended to enhance hydrophobic interactions with guest molecules while preserving the native quaternary architecture. Temperature-dependent changes in surface hydrophobicity and solvent accessibility were probed using the environment-sensitive fluorescent dye ANS, enabling a comparative assessment of the conformational behavior of the two mutants. Overall, this study highlights how targeted modulation of the internal cavity composition of ferritin can tune both its physicochemical properties and stability, providing insights relevant for the rational design of ferritin-based nanoplatforms for biomedical applications.