Tadashi Okobira

Molecular Dynamics Studies of Side Chain Effect on the β-1,3-d-Glucan Triple Helix in Aqueous Solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.

β-1,3-d-Glucan Schizophyllan/Poly(dA) Triple-Helical Complex in Dilute Solution

A certain length of poly(deoxyadenylic acid) (dA(X)) can form a novel complex with β-1,3-D-glucan schizophyllan (SPG) with a stoichiometric composition of one dA binding two main chain glucoses. We measured dilute solution properties for the complex with light and small-angle X-ray scattering as well as intrinsic viscosity and found that the complex behaves as a semiflexible rod without branching or cross-linking. We analyzed the data with the wormlike cylinder model, and the chain dimensions and the persistence length for the complexes were consistently determined. The chain flexibility was reduced to almost 25% upon complexation for dA/SPG and to 15% for S-dA/SPG, where S-dA denotes the phosphorothioated DNA analogue. The changes in the molar mass per unit length and the diameter indicated that the helix was elongated or stretched along the axis direction upon the complexation.

Synthesis and characterization of a calix[4]arene amphiphilie bearing cysteine and uniform Au nanoparticle formation templated by its four cysteine moieties.

A novel calix[4]arene amphiphilic molecule, denoted by CCaL3, was synthesized and found to form a spherical micelle consisting of 12 molecules at low pH in aqueous solution. Furthermore, uniform Au nanoparticles with 2.0 nm in diameter were synthesized in aqueous solution on the template consisting of the four cysteines of the upper rim of CCaL3. Asymmetric field flow fractionation coupled with light scattering showed that there was no dispersity in the CCaL3 micellar aggregation number. When AuCl4(-) ions were added into the CCaL3 micelle solution, induced circular dichroism (ICD) appeared, indicating appearance of the structural chirality of the CCaL3/AuCl4(-) complex. A combination of electron microscopy and small-angle X-ray scattering showed that helically coiled bilayer sheets were formed upon addition of AuCl4(-). Subsequent reduction with the amine of cysteine moieties led to uniform Au nanoparticles formation with 2.0 nm in diameter on the micellar plate surface. The nanoparticle size was almost equal to the size of cavity constructed by the four cysteines on the calix[4]arene upper rim, indicating that the growth of Au nanoparticles was spatially controlled by the host-guest interaction between the cysteines and Au.

Platonic Micelles: Monodisperse Micelles with Discrete Aggregation Numbers Corresponding to Regular Polyhedra

The concept of micelles was first proposed in 1913 by McBain and has rationalized numerous experimental results of the self-aggregation of surfactants. It is generally agreed that the aggregation number (Nagg) for spherical micelles has no exact value and a certain distribution. However, our studies of calix[4]arene surfactants showed that they were monodisperse with a defined Nagg whose values are chosen from 6, 8, 12, 20, and 32. Interestingly, some of these numbers coincide with the face numbers of Platonic solids, thus we named them “Platonic micelles”. The preferred Nagg values were explained in relation to the mathematical Tammes problem: how to obtain the best coverage of a sphere surface with multiple identical circles. The coverage ratio D(N) can be calculated and produces maxima at N = 6, 12, 20, and 32, coinciding with the observed Nagg values. We presume that this “Platonic nature” may hold for any spherical micelles when Nagg is sufficiently small.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

X-ray Scattering from Immunostimulatory Tetrapod-Shaped DNA in Aqueous Solution To Explore Its Biological Activity–Conformation Relationship

We carried out synchrotron X-ray scattering experiments from four DNA supermolecules designed to form tetrapod shapes; these supermolecules had different sequences but identical numbers of total base pairs, and each contained an immunostimulatory CpG motif. We confirmed that the supermolecules did indeed form the expected tetrapod shape. The sample that had the largest radius of gyration (Rg) induced the most cytokine secretion from cultured immune cells. Structural analysis in combination with a rigid tetrapod model and an atomic scale DNA model revealed that the larger Rg can be ascribed to dissociation of the DNA double strands in the central connecting portion of the DNA tetrapod. This finding suggests that the biological activity is related to the ease with which single DNA strands can be formed.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.