Higher order assembly of virus-like particles (VLPs) mediated by multi-valent protein linkers.

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.

Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.

BACKGROUND: Destruction 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 FINDINGS: Mice 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/SIGNIFICANCE: The 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.

Janus-like protein cages. Spatially controlled dual-functional surface modifications of protein cages.

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.

Cutting edge structural protein from the jaws of Nereis virens.

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 approximately 38 kDa histidine-rich protein rich in glycine and histidine (approximately 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.

Critical role of zinc in hardening of Nereis jaws.

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.

Halogenated veneers: protein cross-linking and halogenation in the jaws of nereis, a marine polychaete worm.

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.