Poly(vinyl amine)-silica composite nanoparticles: models of the silicic acid cytoplasmic pool and as a silica precursor for composite materials formation

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Using the same organocatalyst for asymmetric synthesis of both enantiomers of glutamic acid-derived Ni(II) complexes via 1,4-additions of achiral glycine and dehydroalanine Schiff base Ni(II) complexes

(S)- and (R)-BIMBOL were efficient PT catalysts of asymmetric Michael addition of prochiral Ni-PBP-Gly (1) to acrylic esters and malonic esters to Ni-PBP-Δ-Ala (2) correspondingly. The salient feature of the catalysis is opposite configurations of Glu prepared via the two paths with BIMBOL of the same configuration and a perspective novel catalytic procedure for the synthesis of Gla derivatives.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

In-silico Assessment of Protein-Protein Electron Transfer. A Case Study: Cytochrome c Peroxidase – Cytochrome c

The fast development of software and hardware is notably helping in closing the gap between macroscopic and microscopic data. Using a novel theoretical strategy combining molecular dynamics simulations, conformational clustering, ab-initio quantum mechanics and electronic coupling calculations, we show how computational methodologies are mature enough to provide accurate atomistic details into the mechanism of electron transfer (ET) processes in complex protein systems, known to be a significant challenge. We performed a quantitative study of the ET between Cytochrome c Peroxidase and its redox partner Cytochrome c. Our results confirm the ET mechanism as hole transfer (HT) through residues Ala194, Ala193, Gly192 and Trp191 of CcP. Furthermore, our findings indicate the fine evolution of the enzyme to approach an elevated turnover rate of 5.47×10⁶ s⁻¹ for the ET between Cytc and CcP through establishment of a localized bridge state in Trp191.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.