Engineering ferritin nanocage to unravel its self-assembly, iron(II) entry, transit and exit

Abstract

Ferritin protein nanocage sequesters toxic free iron and can store up to 4500 atoms in the form of encapsulated insoluble ferrihydrite (Fe2O3 .xH2O) mineral, in almost all organisms [1,3]. A conserved 3- fold ferritin variant D127E, was designed and analyzed to probe relationships between ferroxidase activity of ferritin and ion channel electrostatic potential/protein structure [2]. D127E mutation abolished the iron uptake (ferroxidase activity), which illustrate the crucial role of the electrostatic potential and the size of the eight, cage-penetrating ion channels, around the 3-fold symmetry axes of ferritin protein cages, on ferritin enzyme activity and mineralization [2]. Iron release and its bio-availability are highly essential for different biological processes such as respiration, DNA synthesis etc.[1,3]. Stable ferritin protein nanocage, low solubility of Fe(III) and presence of dissolved O2 limits iron release. Therefore, in order to overcome this problem, we have altered the protein cage and employed a reductive approach to release iron from ferritin nanocages using a combination of physiological reducing agent and suitable electron transfer mediators. We have altered conserved interactions in hydrophobic 4-fold pores to study iron mobilization and cage stability. Increasing negative charge in hydrophobic ferritin 4-fold pore enhanced iron mobilization (Fe 2+ exit) kinetics but lowered cage stability. We have also found that the modification of amino acid residues located only at the external surface of ferritin affects the migration rate in the presence of an applied electric field and successfully synthesized hybrid ferritin by pH dependent unfolding/refolding technique, which we believe to compliment mass spectrometry to characterize large proteins.