BackgroundDestruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage.Methodology/Principal FindingsMice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms.Conclusions/SignificanceThe immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.

SUMMARY Hardening of invertebrate jaws and mandibles has been previously correlated to diverse, potentially complex modifications. Here we demonstrate directly,for the first time, that Zn plays a critical role in the mechanical properties of histidine-rich Nereis jaws. Using nanoindentation, we show that removal of Zn by chelation decreases both hardness and modulus by over 65%. Moreover, reconstitution of Zn yields a substantial recovery of initial properties. Modulus and hardness of Zn-replete jaws exceed those attainable by current engineering polymers by a factor of >3. Zn-mediated histidine cross-links are proposed to account for this enhancement in mechanical properties.

The fang-like jaws of the marine polychaete Nereis Virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn 2+ by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, NVjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an ∼38 kDa histidine-rich protein rich in glycine and histidine (∼36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.

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Mineralized tissues are produced by most living organisms for load and impact functions. In contrast, the jaws of the clam worm, Nereis, are hard without mineralization. However, they are peculiarly rich in halogens, which are associated with a variety of post-translationally modified amino acids, many of which are multiply halogenated by chlorine, bromine, and/or iodine. Several of these modified amino acids, namely dibromohistidine, bromoiodohistidine, chloroiodotyrosine, bromoiodotyrosine, chlorodityrosine, chlorotrityrosine, chlorobromotrityrosine, and bromoiodotrityrosine, have not been previously reported. We have found that the distributions of Cl, Br, and I differ: Cl is widespread whereas Br and I, although not colocalized, are concentrated in proximity to the external jaw surfaces. By using nanoindentation, we show that Br and I are unlikely to play a purely mechanical role, but that the local Zn and Cl concentrations and jaw microstructure are the prime determinants of local jaw hardness. Several of the post-translationally modified amino acids are akin to those found in various sclerotized structures of invertebrates, and we propose that they are part of a cross-linked protein casing.

Two- and three-dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom-up approaches. In this study, higher order assembly of virus-like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer-by-layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi-topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials.

Protein cage architectures, such as viral capsids, ferritins, and heat shock proteins (Hsp), have been extensively used as model systems to study the self-assembly of macromolecular complexes [1][2][3][4][5] and as nanoreactors for materials synthesis. [6][7][8][9][10][11][12][13] However, it is still challenging to manipulate their self-assembly in a controlled way and to analyze their assembled products precisely at the molecular level. 14,15 In this study, we have generated two different individual mutants of a protein cage with functional groups either inside or outside of the cage ( Figure 1A). We chemically modified different cages and reconstructed chimeric cages with a controlled ratio of two subunit types ( Figure 1B). Using mass spectrometry, we were able to determine the compositions of the ensemble population and also of the individual chimeric cages within the population at the molecular level. A model based on a binomial distribution suggested chimeric cages are assembled by random incorporation of the two individual subunits.Mass spectrometry has been used to monitor multicomponent systems, because it can simultaneously resolve individual molecular masses present in a mixture. [16][17][18] Using a combination of electrospray ionization (ESI) 19 and a time-of-flight (TOF) mass analyzer, it is possible to determine the masses of individual protein components of a noncovalently associated macromolecular complex as well as the mass of the intact macromolecular complex without disturbing the structures. 16,[20][21][22][23] The Dps (DNA binding protein from starved cells) from the Gram-positive bacterium Listeria innocua (Li) is a member of the ferritin superfamily and prevents oxidative damage of DNA by accumulating iron atoms within its central cavity to produce an iron oxide core similar to that of ferritins. 24,25 The LiDps consists of 12 identical 18 kDa subunits that self-assemble into a hollow protein cage having tetrahedral 23 symmetry ( Figure 1A). 24 The LiDps has an outer diameter of 9 nm and an inner cavity diameter of 5 nm with 0.8 nm pores at the 3-fold axis where molecules can pass through to the interior ( Figure 1A). 24 The LiDps has been used as a template for nanomaterials synthesis of metal oxides of iron 26 and cobalt 27 as well as cadmium sulfide 28 and platinum 29 with or without modifications. The small number of subunits, robustness at high temperature, 26,27 and intrinsic biomineralizing capability 25 of the LiDps protein cage make it an attractive modifiable nanoreactor for nanomaterials syntheses. In addition, the defined small cavity size 24 allows synthesis of extremely small nanostructured materials. 29 Two individual cysteine mutants, one exposed on the interior surface and the other on the exterior surface, were generated to adapt the LiDps for selective chemical modifications. The serine residue at position 138 located in the middle of helix E where it is directed toward the inside cavity was substituted with cysteine (S138C) 29 ( Figure 1A, blue). Alternatively, for th...

Protein cages have been used both as size-constrained reaction vessels for nanomaterials synthesis and as nanoscale building blocks for higher order nanostructures. We generated Janus-like protein cages, which are dual functionalized with a fluorescent and an affinity label, and demonstrated control over both the stoichiometry and spatial distribution of the functional groups. The capability to toposelectively functionalize protein cages has allowed us to manipulate hierarchical assembly using the layer-by-layer assembly process. Janus-like protein cages expand the toolkit of nanoplatforms that can be used for directed assembly of nanostructured materials.