Simultaneous purification and site-specific modification of pyrroline-carboxy-lysine proteins

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.     

A two-step strategy for structure-activity relationship studies of N-methylated aβ42 C-terminal fragments as aβ42 toxicity inhibitors

Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer's disease. Previously, we found that the C-terminal fragments (CTFs), Aβ(30-42) and Aβ(31-42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x-42) peptides (x=28-39). Therefore, we chose these peptides as leads for further development. These CTFs are short (12-13 amino acids) hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N?terminus resulted in toxic peptides. Therefore, we next incorporated N-methyl amino acids, which are known to increase the solubility of such peptides by disrupting the β-sheet formation. Focusing on Aβ(31-42), we used a two-step N-methyl amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42-induced toxicity. First, each residue was substituted by N-Me-alanine (N-Me-A). In the next step, in positions where substitution produced a significant effect, we restored the original side chain. This strategy allowed exploring the role of both side chain structure and N-Me substitution in inhibitory activity. We found that the introduction of an N-Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31-42). In particular, N-Me amino acid substitution at position 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short peptides is highly structure-specific, providing a basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.     

Abwasserreinigung mit Schilf in einer mobilen Wurzelraumkläranlage

A mobile root‐zone wastewater treatment system with a reaction zone consisting solely of reed roots as the growth surface for microorganisms was tested for temporary applications in the treatment of municipal wastewater. Pretreated municipal wastewater from a large‐scale wastewater treatment plant was fed to this system during the 2016 vegetation period. The outlet limit values for chemical oxygen demand and biochemical oxygen demand were adhered to reliably at an inlet volumetric flow rate of 1200 L d^?1. Short‐term loading peaks of up to 1800 L d^?1 did not lead to overloading of the system.     

Switchable Plasmonic Metasurface Utilizing the Electro-Optic Kerr Effect of a Blue Phase Liquid Crystal

A switchable metasurface composed of plasmonic split ring resonators and a polymer-stabilized liquid crystal blue phase is developed. Owing to field-induced birefringence (electro-optic Kerr effect), the state of polarization of the incident near infrared radiation changes, when a voltage is applied to the liquid crystal. Thus, different resonant modes of the split ring resonators can be addressed and the transmission spectrum changes accordingly. In comparison with other liquid crystal phases, blue phases have several advantages. For example, they are optically isotropic in the field-off state, so that no alignment layer is required. The results of the present study indicate that the advantages of these mesophases can be utilized for switchable metasurfaces.     

A new class of organosilane-modified polycarboxylate superplasticizers with low sulfate sensitivity

It is well-established that a competitive adsorption exists between polycarboxylate superplasticizers (SP) and sulfate ions solubilized in the interstitial solution of cement paste, which may cause a loss of the dispersing properties. This has been explained by competitive weak ionic interactions between functional carboxyl groups and sulfate ions with cement grains. In this study, SPs including trialkoxysilane functional groups have been synthesized. Adsorption and dispersing properties of these SPs were evaluated in the presence of different concentrations of Na₂SO₄ added in aqueous solution. It appeared that a partial substitution of carboxyl groups by trialkoxysilane in the polymer makes them more resistant to sulfate ions. We suggested that the high adsorption capacity of these SPs results from the formation of strong bonds between hydroxysilane groups and calcium silicate hydrate phases. The improved compatibility of these new silylated SPs has been demonstrated through the formulation of two different concrete equivalent mortars.     

Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active‐Site Mutant Library

A deeper understanding of the >99 % S‐selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active‐site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)‐citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)‐citral it remained ≥88 % (S) for all single‐residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A‐based double‐mutant library that enhanced the (E)‐citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)‐citral to 44 % (S). Formation of (R)‐citronellal from an (E/Z)‐citral mixture is a desire in industrial (?)‐menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.     

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate‐2‐Kinase

The efficient synthesis of pure d ‐glycerate‐2‐phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one‐step biocatalytic phosphorylation of glyceric acid. Glycerate‐2‐kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate‐2‐kinase‐catalyzed phosphorylation was followed by ³¹P NMR and showed excellent enantioselectivity towards phosphorylation of the d ‐enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d ‐glycerate 2‐phosphate. This phosphorylation reaction, using recombinant glycerate‐2‐kinase, yielded d ‐glycerate 2‐phosphate in fewer reaction steps and with higher purity than chemical routes.     

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants

Squalene–hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl–farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products.     

Organometallic DNA–B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins

The synthesis and structural characterization of Co-(dN)₂₅-Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)₃₉-Cbl, which are organometallic DNA–B₁₂ conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA–Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA–Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA–Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)₂₅-Cbl, or radiolabeled vitamin B₁₂ (⁵⁷Co-CNCbl) and Co-(dN)₂₅-Cbl or Co-(dN)₃₉-Cbl. Binding of the new DNA–Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA–Cbl, Co-(dN)₁₈-Cbl. The contrasting affinities of TC versus IF and HC for the DNA–Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA–Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA–Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.     

Porous silicon bulk acoustic wave resonator with integrated transducer

ABSTRACT: : We report that porous silicon acoustic Bragg reflectors and AlN-based transducers can be successfully combined and processed in a commercial solidly mounted resonator production line. The resulting device takes advantage of the unique acoustic properties of porous silicon in order to form a monolithically integrated bulk acoustic wave resonator.