Mechanism On One-and Two-Dimensional Self-Assembly Of Soybean Seed Ferritin Induced By Poly (α, L-lysine)

Self-assembly is a ubiquitous phenomenon in the living system, and it is one of the most essential content in life science. The phenomenon of self-assembling of widespread molecules such as proteins, DNA, and peptides in nature is the hotspot of bio-nanotechnology research. Compared with DNA and peptides, the research level of self-assembly of protein biomolecules is much lower due to its complexity of the spatial structures, and the disorder and great amount of of non-covalent interactions. Thus, how to control the non-covalent interactions, especially the electrostatic force, to prepare ordered protein self-assembly is a hotspot of the current study. Ferritins are members of the superfamily of iron storage and detoxification proteins found throughout the animal, plant, and microbial kingdoms. All ferritins are composed of24structurally identical subunits that assemble into a very robust protein cage with octahedral (432) symmetry. The external diameter of these assembled protein cages is12nm and the internal cavity is8nm. Each subunit of the ferritin usually consists of four helix bundle (A, B, C, and D), and a short E helix located at the C-terminus. Different from mammal fertitin, phytoferritin only contains H-type subunit. Particularly, mature soybean seed ferritin (mSSF) is comprised with H-1(26.5kDa) and H-2(28.0kDa) subunits with a high homology. In mSSF, the E-helix of H-1(26.5kDa) subunit is removed during its early configuration, thereby forming a protein which has expanded4-fold channels. In view of two important properties of mSSF structure, a highly negative charge density on the inner cavity under physiological conditions and the expanded symmetrical4-fold channels, we synthesized the reconstructed mSSF (rmSSF) in vitro. In addition, based on the the structural characteristics of mSSF, we designed a linear poly (a, L-lysine)(PLL) to induce ordered self-assembly of rmSSF and explored the polymerization mechanism. The study will broaden the range of applications of ferritin nanotechnology, and provide a good template for the preparation of novel nanomaterials and devices. Main results were bbtained as follows:1. mSSF is a heteropolymer consisting of H-1ΔAE and H-2in a1:1ratio, forming a hollow and spherical structure. Through molecular biology and chemical means, using the reversible assembly property of ferritin, reconstructed mSSF (rmSSF) was prepared. The4-fold channel size of rmSSF was1.2nm in length and0.4nm in width, which was obviously larger than that of its analogue, rH-2, a homopolymer. In addition, this ferritin maintained its hollow and spherical structure and iron oxidiase activity.2. Poly (a, L-lysine)(PLL) is a good example of a water-soluble polymer with positive charges based on a naturally occurring amino acid monomer lysine. PLL with polymerization degree of15(PLL15) was designed, and rmSSF was induced into linear chains in the presence of PLL15through channel-directed electrostatic interaction, and their binding ratio was1:1(PLL15/rmSSF). Moreover, the self-assembly of rmSSF induced by PLL15could be controlled by reacton time and PLL15/ferritin ratio. The pH, ionic strength, and peptide types were also influencing factors. This study demonstrated that the electrostatic force could be controlled to fabricate the hierarchical assembly of supramolecular protein cages.3. To brodern the hierarchical assembly of ferritin cages, urea with low concentrations was used to expand the local position of the4-fold channel by taking advantage of the flexibility of protein channels. The initial rate of iron release (vo) influenced by urea was detected;10.0mM of urea was chosen to expand the4-fold channel of rmSSF. We found that rmSSF could self-assemble into2D square arrays through channel-directed electrostatic interactions with PLL15at pH7.0in the presence of urea, and the PLL15-ferritin binding ratio was3:1. Structurally, protein cages were aligned along their common4-fold symmetry axis, imposing a fixed disposition of neighboring ferritins. To explore the application of this strategy for positioning inorganic nanomaterial, reconstituted holo rmSSF_H193E/H197E with a loading of600iron atoms per shell was prepared, followed by treatment with PLL15in a3:1ratio. It revealed that the resulted Fe (Ⅲ) cores within ferritins arrayed regularly with distances of about10.0nm between centers of neighboring component, in accordance to the protein lattices. Thus, the thinking behind this strategy was that the inner cavity of apoferritin could provide an ideal, spatially restricted, and chemical reaction chamber within which nanoparticles could be accurately positioned. Such2D assembly can be utilized as a scaffold for various functionalities by manipulating three distinct interfaces (the exterior surface, the interior surface, and the interface between subunits) of each protein cage.