Fabrication of Nanoreaction Clusters with Dual-Functionalized Protein Cage Nanobuilding Blocks.

Fabrication of functional nanostructures is a prominent issue in nanotechnology, because they often exhibit unique properties that are different from the individual building blocks. Protein cage nanoparticles are attractive nanobuilding blocks for constructing nanostructures due to their well-defined symmetric spherical structures, polyvalent nature, and functional plasticity. Here, a lumazine synthase protein cage nanoparticle is genetically modified to be used as a template to generate functional nanobuilding blocks and covalently display enzymes (ß-lactamase) and protein ligands (FKBP12/FRB) on its surface, making dual-functional nanobuilding blocks. Nanoreaction clusters are subsequently created by ligand-mediated alternate deposition of two complementary building blocks using layer-by-layer (LbL) assemblies. 3D nanoreaction clusters provide enhanced enzymatic activity compared with monolayered building block arrays. The approaches described here may provide new opportunities for fabricating functional nanostructures and nanoreaction clusters, leading to the development of new protein nanoparticle-based nanostructured biosensor devices.

Analysis of nano-crystals: Evaluation of heavy metal-embedded biological specimen by high voltage electron microscopy.

Heavy metal compounds are adsorbed onto biological specimen in order to enhance the contrast as well as to preserve the structural features of the specimen against electron beam-induced radiation damage. In particular, in combination with computational image processing, negative staining is widely used for structural analysis of protein complexes to moderate resolutions. Image analysis of negatively stained biological specimen is known to suffer from limited achievable resolution due to dehydration and large grain size of staining molecules although the extent of such effect remains somewhat dubious. Stain molecules exist as grains under electron beam. However, clear observation of the crystalline nature of the grains and their association with biological specimen has not been thoroughly demonstrated. In this study, we attempted high-resolution TEM (HRTEM) using high voltage electron microscopy and electron crystallography analysis for the detailed characterization of negatively stained biological specimen, focusing on physical state and chemical composition of the stain molecules. The electron crystallography analysis allowed for the identification of the crystal constituents of widely used stains, hence revealing the chemical nature and the morphology of the stain molecules at specimen level. This study re-evaluated generally accepted notions on negative staining, and may help correctly interpreting the structural analysis of stained biological specimen.

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish.

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.

The actin bundling activity of actin bundling protein 34 is inhibited by calcium binding to the EF2.

Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.

Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue.

The selective detection of specific cells of interest and their effective visualization is important but challenging, and fluorescent cell imaging with target-specific probes is commonly used to visualize cell morphology and components and to track cellular processes. Multiple displays of two or more targeting ligands on a polyvalent single template would make it possible to construct versatile multiplex fluorescent cell imaging probes that can visualize two or more target cells individually without the need for a set of individual probes. To achieve this goal, we used encapsulin, a new class of protein cage nanoparticles, as a template and implanted dual targeting capability by presenting two different affibody molecules on a single encapsulin protein cage nanoparticle post-translationally. Encapsulin was self-assembled from 60 identical subunits to form a hollow and symmetric spherical structure with a uniform size. We genetically inserted SpyTag peptides onto the encapsulin surface and prepared various SpyCatcher-fused proteins, such as fluorescent proteins and targeting affibody molecules. We successfully displayed fluorescent proteins and affibody molecules together on a single encapsulin in a mix-and-match manner post-translationally using bacterial superglue, the SpyTag/SpyCatcher ligation system, and demonstrated that these dual functional encapsulins can be used as target-specific fluorescent cell imaging probes. Dual targeting protein cage nanoparticles were further constructed by ligating two different affibody molecules onto the encapsulin surface with fluorescent dyes, and they effectively recognized and bound to two individual targeting cells independently, which could be visualized by selective colors on demand.

Outer membrane vesicles from ß-lactam-resistant Escherichia coli enable the survival of ß-lactam-susceptible E. coli in the presence of ß-lactam antibiotics.

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from ß-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental ß-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried ß-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from ß-lactam-induced death and/or directly degrade ß-lactam antibiotics. Several proteins that can be involved in degrading ß-lactam antibiotics were more abundant in OMVs from ß-lactam-resistant E. coli, and thus OMVs from ß-lactam resistant E. coli could directly and dose-dependently degrade ß-lactam antibiotics and fully rescue ß-lactam-susceptible E. coli and other bacterial species from ß-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from ß-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against ß-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.