Akiyoshi Tanaka

Structural and thermodynamic analyses of solute-binding Protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I.

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Galβ1–3GlcNAc) and galacto-N-biose (GNB; Galβ1–3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with Kd values of 0.087 and 0.010 μm, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.

Age-Dependent Manganese Hyperaccumulation in Chengiopanax sciadophylloides (Araliaceae)

ABSTRACT Chengiopanax sciadophylloides is a Japanese tree previously known to accumulate large concentrations of manganese (Mn) in its leaves. To date, this distinctive characteristic has not been fully clarified to use these characteristics for physiological models for research of mineral acquisition and phytoremediation of Mn. In this investigation, the accumulation of Mn in both small young and tall mature C. sciadophylloides trees, and its overall distribution through young trees were examined. The foliar Mn concentrations of mature trees were consistently found to exceed 10,000 mg kg− 1 DW, with recorded maximum values of up to 23,500 mg kg− 1, while those of young trees were found to be around 8,700 mg kg− 1. The concentrations of eleven other metals in C. sciadophylloides was also measured, and found that concentration of each divalent cation was lower than average values of many samples from wide area in Japan.

Thermodynamic Effects of Disulfide Bond on Thermal Unfolding of the Starch-Binding Domain of Aspergillus niger Glucoamylase

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with β-cyclodextrin at 25 °C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t 1/2 of the two mutant proteins decreased by about 10 °C as compared to the wild-type protein at pH 7.0. At t 1/2 of the wild-type protein (52.7 °C), the mutant proteins destabilized by about 10 kJ mol−1 in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin‐type prostaglandin (PG)u2003D synthase (L‐PGDS) is a dual‐functioning protein in the lipocalin family, acting as a PGD2‐synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant‐induced unfolding of L‐PGDS follows a four‐state pathway, including an activity‐enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L‐PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L‐PGDS was a completely reversible process at pHu20034.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L‐PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two‐state unfolding mechanism, the ratio of van’t Hoff enthalpy (ΔHvH) to calorimetric enthalpy (ΔHcal) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three‐state unfolding process were performed. The heat capacity curves fit well with a three‐state process; and the estimated transition temperature (Tm) and enthalpy change (ΔHcal) of the N↔I and I↔U transitions were 48.2u2003°C and 190u2003kJ·mol−1, and 60.3u2003°C and 144u2003kJ·mol−1, respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290u2003nm revealed that L‐PGDS unfolded through the intermediate state, where its main chain retained the characteristic β‐sheet structure without side‐chain interactions.

Contributions of polysaccharide and lipid regions of lipopolysaccharide to the recognition by spike G protein of bacteriophage Φx174

A histidine-tagged G protein of bacteriophage φX174 (HisG) bound strongly with lipopolysaccharide (LPS) of Escherichia coli C, one of a φX174-sensitive Ra strain. The dissociation constant, K d, was measured to be 0.16±0.04 μM by fluorometric titration. HisG showed slightly less affinity to LPSs of the insensitive Rc and Rd2 strains having shorter R-core polysaccharide sequences than that of the sensitive Ra strains. The difference between the two types of LPS was demonstrated by CD spectra; LPSs of the sensitive strains increased the signal intensity for β-sheet, while the insensitive strains decreased it. The chemically degraded LPS derivatives lacking a hydrophobic lipid region showed much less affinity to HisG, indicating the importance of the lipid region of LPS for strong binding with HisG. On the other hand, since the degraded derivatives increased the intensity of CD spectra, the polysaccharide region is thought to contribute to the conformation change of the protein.

Cloning and characterization of phytochelatin synthase from a Nickel hyperaccumulator Thlaspi japonicum and its expression in yeast

Abstract Phytochelatin {γ-(EC) n G} is a group of peptides that chelate and detoxify heavy metals in plants. However, the relationship between the phytochelatin function and metal-hyperaccumulating plants has not been elucidated. In the current study, we cloned the phytochelatin synthase (PCS) gene, designated as TjPCS, from a Ni-hyperaccumulator plant Thlaspi japonicum, and examined its ability to detoxify Cd2+. The RT-PCR product of 1,745 bp, which contained the open reading frame of the TjPCS was isolated, and cloned. The deduced amino acid sequence of TjPCS (475 amino acid length) showed a 90% identity with the sequence of PCS from Arabidopsis thaliana. The yeast transformed with the TjPCS produced PCS and showed an obvious Cd2+ tolerance after the induction of gene expression by galactose.

Thermal Unfolding and Modular Architecture of Clostridium stercorarium Xyn10B

To examine the possibility of module interaction in the thermal unfolding of different modular architectures, four truncated proteins were constructed from Clostridium stercorarium Xyn10B: a family 10 catalytic module (CM10), a polypeptide compound of one family 22 carbohydrate-binding module (CBM22-2) and the catalytic module (CBM22-CM10), two family 22 CBMs and the catalytic module (2CBM22-CM10), and only two family 22 CBMs (2CBM22). Thermal unfolding of four proteins were observed by differential scanning calorimetry. CM10 was unfolded reversibly and denatured as one component. The unfolding of protein CBM22-CM10 comprising CBM22-2 connected with CM10 was irreversible, and can be assumed to be one-component denaturation. Protein 2CBM22, with two CBM22s in tandem, unfolded as two independent modules. However, 2CBM22-CM10, with two CBM22s, unfolded as two and not the expected three separate components. These findings constitute the first reported case in which differences in thermal unfolding units and mechanisms were derived from differences in the modular architectures of proteins.

Enhanced Growth and nodule occupancy of red kidney bean and soybean inoculated with soil aggregate-based inoculant

Abstract Volcanic ash soil, which is widely distributed in Japan, contains a large amount of well-structured soil aggregates. By using these aggregates as carrier materials, we prepared (brady)rhizobial inoculants for red kidney bean (Phaseolus vulgaris) and soybean (Glycine max). Autoclaved soil aggregates were inoculated with Rhizobium tropici CIATS99R or Bradyrhizobium japonicum USDA110R, incubated for 15 or 21 d at 30°C, slowly air-dried at 20°C to prepare the aggregate-based inoculants, and stored at various temperatures. The populations of CIATS99R and USDA110R in the aggregate-based inoculants were maintained during several months of storage at 20°C. When the aggregate-based inoculants were mixed with soil, CIATS99R and USDA110R cells showed a remarkably improved survival in soils compared with those mixed with soil without carrier material. The effect of the aggregate-based inoculants on the growth of red kidney bean and soybean was examined in pot experiments. By placing a small amount of the inoculant just beneath the seeds at the time of sowing, plant growth was significantly enhanced compared with the use of traditional peat-based inoculant. In addition, nodule formation on the upper part of soybean roots and nodule occupancy by the inoculated strain were remarkably enhanced by the aggregate-based inoculant. It is suggested that soil aggregates might be suitable carrier materials for preparing cheap and effective (brady)rhizobial inoculants.

Calorimetric studies of the growth of anaerobic microbes

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C.xa0acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method.

Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques

Protein folding is a thermodynamic process driven by energy gaps between the native and unfolded states. Although a wealth of information is available on the structure of folded species, there is a paucity of data on unfolded species. Here, we analyzed the structural properties of the unfolded state of the starch-binding domain of glucoamylase from Aspergillus niger (SBD) formed in the presence of guanidinium hydrochloride (GuHCl). Although far-UV CD and intrinsic tryptophan fluorescence spectra as well as small angle X-ray scattering suggested that SBD assumes highly unfolded structures in the presence of GuHCl, near-UV circular dichroism of wild-type SBD suggested the presence of residual structures in the unfolded state. Analyses of the unfolded states of tryptophan mutants (W543L, W563A, W590A and W615L) using Similarity Parameter, a modified version of root mean square deviation as a measure of similarity between two spectra, suggested that W543 and W563 have preferences to form native-like residual structures in the GuHCl-unfolded state. In contrast, W615 was entirely unstructured, while W590 tended to form non-native ordered structures in the unfolded state. These data and the amino acid sequence of SBD suggest that local structural propensities in the unfolded state can be determined by the probability of the presence of hydrophobic or charged residues nearby tryptophan residues.