Conversion of the Native 24‐mer Ferritin Nanocage into Its Non‐Native 16‐mer Analogue by Insertion of Extra Amino Acid Residues

Abstract

Protein assemblies with high symmetry are widely distributed in nature.M ost efforts so far have focused on repurposing these protein assemblies,astrategy that is ultimately limited by the structures available.T oo vercome this limitation, methods for fabricating novel self-assembling proteins have received intensive interest. Herein, by reengineering the key subunit interfaces of native 24-mer protein cage with octahedral symmetry through amino acid residues insertion, we fabricated a1 6-mer lenticular nanocage whose structure is unique among all knownprotein cages.This newly non-native protein can be used for encapsulation of bioactive compounds and exhibits high uptake efficiency by cancer cells. More importantly,the abovestrategy could be applied to other naturally occurring protein assemblies with high symmetry, leading to the generation of new proteins with unexplored functions.Livingsystemsutilizeproteinsasbuildingblockstoconstruct alarge variety of self-assembled nanoscale architectures.The assembly mechanism is governed by noncovalent interactions, such as hydrogen bonding,h ydrophobic effects and van der Waals interactions.Although separately these interactions are rather weak and transient, when am ultitude of noncovalent bonds act synergistically,s table protein architectures are shaped. [1][2][3] In any self-assembling protein architecture,s ubunit-subunit interactions (SSIs) play an important role.These noncovalent SSIs form large,h ighly complementary,l owenergy subunit-subunit interfaces.S uch interfaces spontaneously self-assemble and allow precise definition of the orientation of subunits relative to one another. Reported approaches to designing self-assembling proteins have satisfied this requirement in different ways,s uch as the use of engineered disulfide bonds, [4,5] electrostatic interactions, [6] chemical cross-links, [7] metal-mediated interactions, [8] ligandinduced association, [9] computational interface design, [10,11] or genetic fusion of multiple protein domains or fragments. [12,13] Theability to control SSIs would lead to the fabrication of the novel nanoscale protein materials with unexplored properties. However,t he task of rendering SSIs controllable is complicated by the fact that SSIs are mediated by weak, noncovalent interactions over large surfaces. [14,15] Protein cages are widely distributed in nature,w hich are constructed from as mall number of subunit building blocks by virtue of their subunit surfaces that self-assemble to highly cooperative,symmetrical structures.Incells,these cages have al arge variety of functions.F or example,v iral capsids are involved in nucleic acid storage and transport, [16] clathrin cages in endocytosis, [17] carboxysomes in CO 2 fixation, [18] Dps in DNAp rotection, [19] and ferritin cages in iron metabolism. [20] These protein cages have recently attracted considerable attention from researchers in the field of nanoscience and nanotechnology because they possess many useful properties such as high symmetry,s olubility and stability, monod...

The Size Flexibility of Ferritin Nanocage Opens a New Way to Prepare Nanomaterials

“…Indeed, the gallic acid‐loaded ferritin sample exhibits a similar color to free gallic acid sample, while apoferritin alone has no color (Figure b). Analytical ultracentrifugation revealed that apo rHuHF sedimented as a single discrete species with s 20,w = 18.9 ± 0.1 S (Figure S6, Supporting Information), agreeing with a reported value . As expected, the gallic acid‐loaded ferritin exhibits nearly the same sedimentation coefficient as apo ferritin alone, indicating that ferritin is still a 24‐mer oligomer after the above encapsulation process.…”

Acute Toxicity of Divalent Mercury to Bacteria Explained by the Formation of Dicysteinate and Tetracysteinate Complexes Bound to Proteins in Escherichia coli and Bacillus subtilis

“…Details of the experimental methods are given in section S1 of the Supporting Information (SI). 15,17 Experiments were performed with the E. coli TOP10 strain (genotype in SI, section S2) and the B.…”

“…The exposure protocol was the same as the one described previously for Pseudomonas idrijaensis. 15 Briefly, a bacterial biomass reaching 1 OD600 unit (corresponding to 0.3 g/L bacteria) was prepared in LB medium at 37°C, then centrifuged and washed in minimally complexing medium (MCM buffer). The composition of the MCM buffer was 20 mM 3-(Nmorpholino)propane sulfonic acid (MOPS buffer), 0.41 mM MgCl2, 6 mM (NH4)2SO4, and 10 mM glucose.…”

“…14 Because almost all mercury trafficking proteins bind Hg linearly with two cysteine groups, mercury-resistant bacteria endowed with the mer operon should contain more Hg(CysR)2 than mercury-sensitive microbes. Using high energy-resolution X-ray absorption near-edge structure (HR-XANES) spectroscopy, it was shown recently 15 that Pseudomonas idrijaensis, which carries the merA locus, contained 56% more Hg(CysR)2 than Escherichia coli and Bacillus subtilis exposed to 20 nM Hg as HgCl2 (intracellular [Hg] = 4.3 ± 0.2 mg Hg/kg dry weight, dw). A minor tetrahedral species attributed to a tetrathiolate complex (Hg(CysR)4) also was detected in the three bacteria.…”

Precision engineering of targeted nanocarriers

“…Ferritin is composed of 24 subunits that self‐assemble into 12 nm cages. The size of the cages can be increased to 16 nm (48 subunits) or decreased to 10 nm (16 subunits) by genetic manipulation (Zhang et al, ; Zhang et al, ). These different sizes may be favorable depending on the intended application.…”