Design of a Hierarchical Assembly at a Solid-Liquid Interface Using an Asymmetric Protein Needle.

Design and control of processes for a hierarchical assembly of proteins remain challenging because it requires consideration of design principles with atomic-level accuracy. Previous studies have adopted symmetry-based strategies to minimize the complexity of protein-protein interactions and this has placed constraints on the structures of the resulting protein assemblies. In the present work, we used an anisotropic-shaped protein needle, gene product 5 (gp5) from bacteriophage T4 with a C-terminal hexahistidine-tag (His-tag) (gp5_CHis), to construct a hierarchical assembly with two distinct protein-protein interaction sites. High-speed atomic force microscopy (HS-AFM) measurements reveal that it forms unique tetrameric clusters through its N-terminal head on a mica surface. The clusters further self-assemble into a monolayer through the C-terminal His-tag. The HS-AFM images and displacement analyses show that the monolayer is a network-like structure rather than a crystalline lattice. Our results expand the toolbox for constructing hierarchical protein assemblies based on structural anisotropy.

Protein Needles Designed to Self-Assemble through Needle Tip Engineering.

The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of ß-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein-protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

Development of Low-Frequency Phased Array for Imaging Defects in Concrete Structures.

The nondestructive inspection of concrete structures is indispensable for ensuring the safety and reliability of aging infrastructures. Ultrasonic waves having a frequency of tens of kHz are frequently used to reduce the scattering attenuation due to coarse aggregates. Such low frequencies enable the measurement of the thickness of concrete structures and detection of layer-type defects, such as delamination, whereas it causes a lack of sensitivity to crack-type defects. In this paper, to realize the ultrasonic phased array (PA) imaging of crack-type defects, we fabricated a low-frequency (LF) array transducer with a center frequency of hundreds of kHz. To avoid the crosstalk between piezoelectric elements and dampen the vibration of each element, we adopted soft lead zirconate titanate (soft PZT) with a low mechanical quality factor. Subsequently, we optimized the geometry of each piezoelectric element using a finite element method to generate a short pulse. After validating the design in a fundamental experiment using a single-element transducer, we fabricated a 32-element array transducer with a center frequency of 350 kHz. To show the imaging capability of the LF array transducer, we applied it to a concrete specimen with a delamination. As a result, the PA with the LF array transducer clearly visualized the delamination, which could not be visualized using the PA with a 2.5 MHz array transducer. Furthermore, we applied it to a more challenging defect, a slit, which is sometimes used to simulate crack-type defects. As a result, the PA with the LF array transducer clearly visualized a slit of 1 mm width and 40 mm height in a concrete specimen. Thus, we demonstrated the usefulness of the LF array transducer for inspecting crack-type defects.

The Versatile Manipulations of Self-Assembled Proteins in Vaccine Design.

Protein assemblies provide unique structural features which make them useful as carrier molecules in biomedical and chemical science. Protein assemblies can accommodate a variety of organic, inorganic and biological molecules such as small proteins and peptides and have been used in development of subunit vaccines via display parts of viral pathogens or antigens. Such subunit vaccines are much safer than traditional vaccines based on inactivated pathogens which are more likely to produce side-effects. Therefore, to tackle a pandemic and rapidly produce safer and more effective subunit vaccines based on protein assemblies, it is necessary to understand the basic structural features which drive protein self-assembly and functionalization of portions of pathogens. This review highlights recent developments and future perspectives in production of non-viral protein assemblies with essential structural features of subunit vaccines.

Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy.

Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.