Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering.

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.

Conformation of poly(methacrylic acid) in acidic aqueous solution studied by small angle X-ray scattering.

The nature of the contracted form of poly(methacrylic acid) PMA chain in salt-free acidic aqueous solution was studied by analyzing scattering curves registered by small-angle X-ray scattering, comparing it with those of PMA in methanol at 26 degrees C and of partially neutralized PMA in aqueous solution containing added salt (the concentration of added salt, Cs=0.1 M NaF). It is shown that the distribution of segments in the contracted form as well as that of PMA in methanol is that of a random-coil in a theta medium and that this distribution of segments is stable over a fair range of degrees of ionization alpha for Cs below 0.1 M. Moreover, the persistence length of PMA at Cs=0.1 M (4+/-0.5 A) is substantially constant throughout the entire range of alpha, indicating that the contracted form of PMA changes to an expanded random-coil in a higher pH region without a significant change in the chain flexibility.

Applicability of the potentiometric titration method to the analysis of the expansion process of bovine plasma albumin in acidic solutions.

To study the expansion process of bovine plasma albumin in acidic solutions, observed potentiometric titration curves at three different ionic strengths were compared with theoretical curves, using the radii of the protein determined by small angle X-ray scattering (SAXS). From the comparison, it was concluded that the expansion is completed via two different transitions and that the conformation of the protein before the first transition is stable and common at all ionic strengths, whereas the form of the protein becomes a more swollen and unstable one after the first transition. Moreover, the charge-independent part of the standard free energy change, delta G0, in the first transition was estimated from the potentiometric titration curves. The numerical value of delta G0 is 2350 +/- 50 cal/mol, which is very small compared with the corresponding one for ordinary biopolymers.

Aggregation of bovine serum albumin upon cleavage of its disulfide bonds, studied by the time-resolved small-angle X-ray scattering technique with synchrotron radiation.

A rapid mixing system of the stopped-flow type, used with small-angle X-ray scattering equipment using synchrotron radiation, is described. The process of aggregation of bovine serum albumin was traced with a time interval of 50 s, initiated upon cleavage of its disulfide bonds by reduction with dithiothreitol. The results indicate that a 218-fold molar excess of dithiothreitol over the number of moles of disulfide bonds in bovine serum albumin is sufficient to initiate the reaction immediately after mixing, which reaches equilibrium in about 15 min. On the other hand, half this amount is not sufficient to initiate the reaction, so that the reaction is delayed by about 150 s. Such a single-shot time-resolved experiment showed that experiments with a time interval of 100 ms are possible with repeated multi-shot runs.

Small angle X-ray scattering studies on local structure of tobacco mosaic virus RNA in solution.

Effects of temperature and ionic strength (S) on the local structure of tobacco mosaic virus RNA in phosphate buffer solution are studied by analyzing the small-angle X-ray scattering (SAXS) curves. The root-mean-square radius of a cross-section of RNA chain was kept at 0.845+/-0.005 nm over a wide range of S from 0.2 to 0.003 at 20 degrees C, whereas it gradually diminished from 0.85 to 0.61 nm when the temperature is raised from 20 to 50 degrees C at S = 0.2. Nevertheless, all of SAXS curves reflecting the backbone structures were equally mimicked by theoretical ones of freely hinged rod (FHR) models, i.e. several straight rods joined with freely hinged joints in the form of a combination of the letter Y, if the constituent rod lengths in the models are adjusted. From these facts, it is suggested that the local structure of the RNA chain in aqueous solution is characterized by an essential feature that unpaired bases in the partially double-stranded helix are constantly far isolated from each other along the helix and the rod-like structure of the helix is preserved over a range of helical contents. Such a characteristic local structure of the chain is entirely collapsed in the formamide solution at 50 degrees C.

Derivation of the small-angle x-ray scattering functions for local conformations of polypeptide chains in solution.

The small-angle x-ray scattering (SAXS) functions are analytically derived for both the randomly coiled and helical local conformations of a polypeptide chain in solution. The resulting scattering functions for helices of various types are characterized by a maximum in the range of scattering-vector corresponding to Bragg spacings of 3-5 A, whereas the random-coil function has no maximum. This result is compatible with the extant SAXS data for partially neutralized poly(L-glutamic acid) and poly(L-lysine) in aqueous solutions. Comparison of the SAXS data with the calculated scattering functions shows that helical structures in both polypeptide chains are of the 3.6(13)-helix (alpha-helix) rather than 3.0(10)-type.

Applicability of broken-rodlike chain model to conformational analysis of polypeptide chain.

In order to check the applicability of the broken-rodlike (BR) chain model, consisting of several rods alternatively joined by flexible random coils, to the conformational analysis of a polypeptide chain in the helix-to-coil transition regions, two relations predicted by the Zimm and Bragg theory and the method with the BR chain model are compared. It is shown that, despite a clear difference between the models employed in the two methods, they give substantially identical results in both probability P(j) that a helical residue is in a helical sequence j units long and averaged helical fraction dependence of the mean-squared radius of gyration. Thus the use of the method with the BR chain model in the conformational analysis of a polypeptide chain could be rationalized, at least, with the same degree of approximation as is assumed in the Zimm and Bragg theory. Using the scattering function for the BR chain model, averaged helical-sequence lengths are evaluated for partially ionized poly(L-glutamic acid) (PGA) in added-salt aqueous solution and nonionized PGA in N-methylacetamide, both in a helical state. As a result, it is shown that the length in the latter molecule is approximately tenfold longer than that in the former one.

Roseigium denhamense gen. nov., sp. nov. and Roseibium hemelinense sp. nov., aerobic bacteriochlorophyll-containing bacteria isolated from the east and west coasts of Australia.

Phenotypic and phylogenetic studies were performed with 10 strains of bacteriochlorophyll-containing bacteria isolated from a variety of marine environments (surface of Rhodophyta, sand and algal sand mat) on the east and west coasts of Australia. The strains were aerobic, chemoheterotrophic, Gram-negative, motile rods with peritrichous flagella. Bacteriochlorophyll a was synthesized under aerobic conditions. Catalase, nitrate reductase, oxidase and phosphatase were produced. ONPG reaction was positive. The strains have been divided into genotype group 1 (seven strains) and genotype group 2 (three strains) according to previously described DNA-DNA hybridization data. Strains OCh 254T and OCh 368T have been included in genotype groups 1 and 2, respectively. The results of 165 rRNA gene sequence comparisons revealed that strains OCh 254T and OCh 368T formed a new cluster within the alpha-2 group of the alpha subclass of the Proteobacteria. The similarity value of the 16S rRNA gene sequences between strain OCh 254T and the most closely related species, Stappia aggregata, was 95.6 %. The sequence similarity value between strains OCh 254T and OCh 368T was 97.1%. It was concluded that these two strains should be placed into a new genus, Roseibium gen. nov., as Roseibium denhamense sp. nov. and Roseibium hamelinense sp. nov. The type species of the genus is Roseibium denhamense. The type strains of Roseibium denhamense and Roseibium hamelinense are OCh 254T (= JCM 10543T) and OCh 368T (= JCM 10544T), respectively.

Roseivivax halodurans gen. nov., sp. nov. and Roseivivax halotolerans sp. nov., aerobic bacteriochlorophyll-containing bacteria isolated from a saline lake.

Phenotypic and phylogenetic studies were performed with two strains (OCh 239T and OCh 210T, T = type strain) of aerobic bacteriochlorophyll-containing bacteria isolated from the charophytes and the epiphytes on the stromatolites, respectively, of a saline lake located on the west coast of Australia. Both strains were chemoheterotrophic, Gram-negative and motile rods with subpolar flagella. Catalase and oxidase were produced. ONPG reaction was positive. Cells utilized D-glucose, acetate, butyrate, citrate, DL-lactate, DL-malate, pyruvate, succinate, L-aspartate and L-glutamate. Acids were produced from D-fructose and D-glucose. Bacteriochlorophyll a was synthesized under aerobic conditions. Strain OCh 239T had nitrate reductase and phosphatase. Acids were produced from L-arabinose, D-galactose, lactose, maltose, D-ribose and sucrose. The strain could grow in 0-20.0% (w/v) NaCl. Strain OCh 210T had urease. Hydrolysis of gelatin was positive. Acids were produced from D-xylose. The strain could grow in 0.5-20.0% (w/v) NaCl. The results of 16S rRNA sequence comparisons revealed that strains OCh 239T and OCh 210T formed a new cluster within the alpha-3 group of the alpha subclass of the class Proteobacteria. The similarity value of the 16S rRNA sequences between strains OCh 239T and OCh 210T was 95.8%. Therefore, it was concluded that these two strains should be placed in a new genus, Roseivivax gen. nov., as the new species Roseivivax halodurans sp. nov. and Roseivivax halotolerans sp. nov. The type species of the genus is Roseivivax halodurans. The type strains of Roseivivax halodurans and Roseivivax halotolerans are OCh 239T (= JCM 10272T) and OCh 210T (= JCM 10271T), respectively.

Modulation of smooth muscle calponin by protein kinase C and calmodulin.

When smooth muscle calponin was incubated with protein kinase C, 1 mole of phosphate was incorporated per mole of calponin. The apparent Km value for calponin of the protein kinase was about 0.4 microM. The phosphorylation of calponin by protein kinase C was inhibited markedly by calmodulin in a calcium-dependent manner. Kinetic analysis of calmodulin-induced inhibition of calponin phosphorylation by protein kinase C revealed that calmodulin inhibited the phosphorylation in a noncompetitive fashion with calponin and the determined Ki value was 0.4 microM. These results suggest that interaction of calmodulin with calponin may play a regulatory role in the phosphorylation by protein kinase C and smooth muscle contraction.

Rubrimonas cliftonensis gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from a saline lake.

Phenotypic and phylogenetic studies were performed with two strains (OCh 317T and OCh 318; T = type strain) of aerobic chemoheterotrophic bacteriochlorophyll-containing bacteria isolated from water of a saline lake located on the west coast of Australia. Both strains were Gram-negative, short rods and were motile by means of polar flagella. Catalase, oxidase, nitrate reductase, phosphatase and urease were produced. The cells utilized D-glucose, citrate, glycolate, pyruvate and ethanol. Acids were produced from L-arabinose, D-fructose, D-galactose, D-glucose, D-ribose and D-xylose. The strains could grow in media containing 0.5-7.5% NaCl. Bacteriochlorophyll a was synthesized under aerobic conditions. The results of 16S rRNA gene sequence comparisons revealed that strain OCh 317T represented a new lineage in the alpha-3 group of the class Proteobacteria. Strains OCh 317T and OCh 318 were identified as strains of the same species because of their very similar phenotypic characteristics and their previously described high DNA-DNA homology. Therefore, it was concluded that the two strains should be assigned to a new genus and species, for which the name Rubrimonas cliftonensis is proposed. The type strain is OCh 317T (= JCM 10189T).