Predicting a viable pH-induced peptide switch to be incorporated with human L-chain ferritin

Ferritin, a ubiquitous iron-storage protein, is an attractive candidate for use in drug delivery systems (DDS) due to its inherently stable cage complex, its ability to encapsulate small molecules, and its genetic manipulability. Although native ferritin presents as a viable drug delivery vehicle, modifications are needed for it to be a well-suited DDS under physiologically relevant conditions. Research indicates that replacing the E-helix of human light chain ferritin with an alternating “Gala” peptide repeat will trigger a pH-induced cage disassembly at a pH below 6. However, despite using published protocols, previous attempts to purify and characterize this modified ferritin in our lab have resulted in confinement of the chimeric protein to insoluble lysate pellets. Thus, further lab experimentation has been limited. This project aimed to propose alternative peptide sequences in silico that could retain pH-switch potential while also enhancing protein solubility. Using Gala as a template, combinations of alanine and leucine residues were substituted in favor of polar amino acids histidine, serine, threonine, asparagine, and glutamine. We used UCSF Chimera to construct the synthetic peptide pdf files, JPred4 to predict secondary structure, and H++ to predict pKa values for comparison with the Gala reference. With the exception of threonine, all of the proposed sequences predicted an -helical secondary structure — with an additional probability (\u3e90%) of adopting a coiled-coil structural motif — and predicted pKa values with 0.25 units of the Gala reference. Based on these results, five attractive E-helix substitutions were selected to clone, purify, express, and evaluate