Tomoya Imai

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitrosizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1→3)-β-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg2+, were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IVI, a disorganized form of cellulose I. Besides these similarities, the in vitromicrofibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitromicrofibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

Mutated Barley (1,3)-beta-D-Glucan Endohydrolases Synthesize Crystalline (1,3)-beta-D-Glucans

Barley (1,3)-β-d-glucan endohydrolases (EC 3.2.1.39), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with α-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with α-laminaribiosyl fluoride and 4′-nitrophenyl β-d-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k cat K m −1) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 × 10−4 m −1 s−1, respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6–8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-β-d-glucans with a degree of polymerization of 30–34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-β-d-glucan chains adopt a parallel, triple helical conformation. The (1,3)-β-d-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-β-d-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-β-d-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-β-d-linked glucosyl residues onto other oligosaccharides or glycoproteins.

Comparative Study of the Gating Motif and C-type Inactivation in Prokaryotic Voltage-gated Sodium Channels

Prokaryotic voltage-gated sodium channels (NaVs) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic NaV, as well as of three new homologues cloned from Bacillus licheniformis (NaVBacL), Shewanella putrefaciens (NaVSheP), and Roseobacter denitrificans (NaVRosD). We found that, although activated by a lower membrane potential, NaVBacL inactivates as slowly as NaChBac. NaVSheP and NaVRosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the “glycine hinge” and “PXP motif” in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic NaVs, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in NaVBacL (A214G), NaVSheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), NaVBacL (G224A), and NaVRosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic NaVs does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

Transformation of peptide nanotubes into a vesicle via fusion driven by stereo-complex formation.

Two types of peptide nanotubes, one is prepared from an amphiphilic peptide having a right-handed helix segment and the other from that having a left-handed helix segment, are shown to transform the morphology into a vesicle by membrane fusion due to stereo-complex formation between these helical segments.

Formation of Highly Twisted Ribbons in a Carboxymethylcellulase Gene-Disrupted Strain of a Cellulose-Producing Bacterium

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Geometric phase analysis of lattice images from algal cellulose microfibrils

A geometric phase analysis has been applied to high-resolution transmission electron microscopy images from algal cellulose microcrystals. The pictures were decomposed into images containing selectively the amplitude or phase information associated to selected Bragg reflections. Compared with Ib (monoclinic)-rich cellulose microfibrils, Ia(triclinic)-rich microfibrils were found to be more heterogeneous when viewed along the H-bonding sheets. As a microfibril twist and radiation damage could not be totally ruled out as having an effect on the lattice image, this result has to be considered with care when used in order to survey the distribution of different allomorphs in a cellulose microfibril. However, the geometric phase analysis of noisy low dose high-resolution images appears as a promising new method to investigate polymer crystals and the distribution of domains having different structures or containing lattice distortions. q 2002 Elsevier Science Ltd. All rights reserved.

Rational design of peptide nanotubes for varying diameters and lengths.

Amphiphilic helical peptides (Sar)m‐b‐(L‐Leu‐Aib)n (m = 22–25; n = 7, 8, 10) with a hydrophobic block as a right‐handed helix were synthesized and their mixtures with (Sar)25‐b‐(D‐Leu‐Aib)6 containing the hydrophobic block as a left‐handed helix were examined in their molecular assembly formation. The single component (Sar)25‐b‐(D‐Leu‐Aib)6 forms peptide nanotubes of 70 nm diameter and 200 nm length. The two‐component mixtures of (Sar)25‐b‐(D‐Leu‐Aib)6 with (Sar)24‐b‐(L‐Leu‐Aib)7, (Sar)22‐b‐(L‐Leu‐Aib)8, and (Sar)25‐b‐(L‐Leu‐Aib)10 yield peptide nanotubes of varying dimensions with 200 nm diameter and 400 nm length, 70 nm diameter and several micrometer length (maximum 30 µm), and 70 nm diameter and 100–600 nm length, respectively. The right‐handed and the left‐handed helix were thus found to be molecularly mixed due to the stereo‐complex formation and to generate nanotubes of different sizes. When the mismatch of the hydrophobic helical length between the two components was of four residues, the longest nanotube was generated. Correspondingly, the hydrophobic helical segments have to interdigitate with an anti‐parallel orientation at the hydrophobic core region of the nanotube. Copyright

Morphology Control between Twisted Ribbon, Helical Ribbon, and Nanotube Self-Assemblies with His-Containing Helical Peptides in Response to pH Change

pH-Responsive molecular assemblies with a variation in morphology ranging from a twisted ribbon, a helical ribbon, to a nanotube were prepared from a novel A3B-type amphiphilic peptide having three hydrophilic poly(sarcosine) (A block) chains, a hydrophobic helical dodecapeptide (B block), and two histidine (His) residues between the A3 and B blocks. The A3B-type peptide adopted morphologies of the twisted ribbon at pH 3.0, the helical ribbon at pH 5.0, and the nanotube at pH 7.4, depending upon the protonation states of the two His residues. On the other hand, another A3B-type peptide having one His residue between the A3 and B blocks showed a morphology change only between the helical ribbon and the relatively planar sheets with pH variation in this range. The morphology change is thus induced by one- or two-charge generation at the linking site of the hydrophilic and hydrophobic blocks of the component amphiphiles but in different ways.

Tubulation on peptide vesicles by phase-separation of a binary mixture of amphiphilic right-handed and left-handed helical peptides

A binary mixture of amphiphilic polypeptides with right- and left-handed helical blocks was self-assembled into a morphology of a nano round-bottom flask shape upon heat treatment. The tubulation occurred as a result of phase-separation into the tubule domain of the single component and the vesicle domain of the stereo-complex.

Directional degradation of β‐chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β‐chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β‐chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β‐chitin microcrystals, and also to label the reducing tips of β‐chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.