Kinetics of Ferritin Self-Assembly by Laser Light Scattering: Impact of Subunit Concentration, pH, and Ionic Strength

Abstract

Ferritins, the cellular iron repositories, are self-assembled, hollow spherical nanocage proteins composed of 24 subunits. The self-assembly process in ferritin generates the electrostatic gradient to rapidly sequester Fe­(II) ions, thereby minimizing its toxicity (Fenton reaction). Although the factors that drive self-assembly and control its kinetics are little investigated, its inherent reversibility has been utilized for cellular imaging and targeted drug delivery. The current work tracks the kinetics of ferritin self-assembly by laser light scattering and investigates the factors that influence the process. The formation of partially structured subunit-monomers/dimers, at pH ≤ 1.5, serves as the starting material for the self-assembly, which upon increasing the pH exhibits biphasic behavior (a rapid assembly process coupled with subunit folding followed by a slower reassembly/reorganization process) and completes within 10 min. The ferritin self-assembly accelerated with subunit concentration and ionic strength (t 1/2 decreases in both the cases) but slowed down with the pH of the medium from 5.5 to 7.5 (t 1/2 increases). These findings would help to regulate the ferritin self-assembly to enhance the loading/unloading of drugs/nanomaterials for exploiting it as a nanocarrier and nanoreactor.

Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages

“…20 One study revealed that ferritin disassembles at an extremely acidic pH 1.5 and then shows a rapid reassembly upon return to neutral pH 7.0, accompanied by folding, followed by a slow phase in which the final 24-mer nanocage is formed. 20 Importantly, this fundamental work led to the rational redesign For each structure, the pentameric and hexameric units are shown in light and saturated colors, respectively. Within the expanded pentameric or hexameric units, the tryptophan (W) residues belonging to individual subunits are highlighted in red: Tm: W19, W48, W70, W90, and W180; Mx: W17, W96, and W155; and Qt: W95 and W154.…”

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production

“…Moderate concentrations of sodium chloride ranging from 0.5 to 1.0 M notably enhanced rhFTL recovery and did not significantly attenuate the removal of impurities at pH 4.4. Recent research also revealed that ionic strength can facilitate ferritin self-assembly in vitro [29,30]. Perhaps this benefit mainly ascribes to the formation of a salt-bridge, which was reported to critically contribute to the physical stability of FTL [28].…”

Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes

“…38 Additionally, ferritin has been found to have a biphasic reassembly, where an initial fast step occurs with folding of subunits and unknown stable intermediates, followed by the slower restructuring of intermediates into the nanocage. 23 Data of pH 13 reassembly rate for Mx-Enc and Qt-Enc was not included, as the presence of aggregation prevented extraction of accurate DLS size values. This suggests that reassembly from the pH 13 disassembled state for the larger and more complex Mx-Enc and Qt-Enc may be prone to protein misfolding pathways and/or partial assembly, and thus, without additional re-engineering, may not be ideal for use in future applications.…”

“…For instance, the disassembly/reassembly of protein nanocages belonging to the ferritin family have been studied via a combination of: intrinsic tryptophan fluorescence (ITF), circular dichroism (CD), UV/VIS spectroscopy and synchrotron small-angle X-ray scattering (SAXS) measurements to assess protein conformation [19][20][21][22] , transmission electron microscopy (TEM) and dynamic light scattering (DLS) to evaluate structural integrity, shape and size distribution, and laser light scattering to monitor assembly kinetics. 23 One study revealed that ferritin disassembles at extremely acidic pH 1.5, then shows a rapid reassembly upon return to neutral pH 7.0, accompanied by folding, followed by a slow phase in which the final 24-mer nanocage is formed. 23 Importantly, this fundamental work led to the rational re-design of ferritin subunit interfaces, resulting in engineered nanocages capable of disassembly at a more amenable pH 4.0.…”

“…23 One study revealed that ferritin disassembles at extremely acidic pH 1.5, then shows a rapid reassembly upon return to neutral pH 7.0, accompanied by folding, followed by a slow phase in which the final 24-mer nanocage is formed. 23 Importantly, this fundamental work led to the rational re-design of ferritin subunit interfaces, resulting in engineered nanocages capable of disassembly at a more amenable pH 4.0. 24, 25 Such modification now permits labile compounds (e.g.…”