Microseeding – A Powerful Tool for Crystallizing Proteins Complexed with Hydrolyzable Substrates

Hydrolysis is an often-encountered obstacle in the crystallization of proteins complexed with their substrates. As the duration of the crystallization process, from nucleation to the growth of the crystal to its final size, commonly requires several weeks, non-enzymatic hydrolysis of an “unstable” ligand occurs frequently. In cases where the crystallization conditions exhibit non neutral pH values this hydrolysis phenomenon may be even more pronounced. ChoX, the substrate binding protein of a choline ABC-importer, produced crystals with its substrate acetylcholine after one month. However, these crystals exhibited only choline, an acetylcholine hydrolysis product, in the binding site. To overcome this obstacle we devised a microseeding protocol leading to crystals of ChoX with bound acetylcholine within 24 hours. One drawback we encountered was the high twinning fraction of the crystals, possibly was due to the rapid crystal growth

The structure of MadC from Clostridium maddingley reveals new insights into class I lanthipeptide cyclases

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC

Easy and Rapid Purification of Highly Active Nisin

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC50 of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure

Technological aspects of gaseous pixel detectors fabrication

Integrated gaseous pixel detectors consisting of a metal punctured foil suspended in the order of 50μm over a pixel readout chip by means by SU-8 insulating pillars have been fabricated. SU-8 is used as sacrificial layer but metallization over uncrosslinked SU-8 presents adhesion and stress problems. In this paper we describe the several methods we have investigated to fabricate a metal layer on top of a partially crosslinked SU-8 film and the challenges we have encountered. The fabrication process using wafer post processing has been proven, but in cases where single chip processing is desirable, edge bead is a major problem to overcome as it can cover a considerable chip area, reducing the detector performance; we show different techniques to reduce this edge bead and improve detection efficiency

The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation

Insights into the Mechanism of Ligand Binding to Octopine Dehydrogenase from Pecten maximus by NMR and Crystallography

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD+, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail

Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis)

Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines

Development and measurement of guidelines-based quality indicators of caesarean section care in the Netherlands: A RAND-modified delphi procedure and retrospective medical chart review

Background There is an ongoing discussion on the rising CS rate worldwide. Suboptimal guideline adherence may be an important contributor to this rise. Before improvement of care can be established, optimal CS care in different settings has to be defined. This study aimed to develop and measure quality indicators to determine guideline adherence and identify target groups for improvement of care with direct effect on caesarean section (CS) rates. Method Eighteen obstetricians and midwives participated in an expert panel for systematic CS quality indicator development according to the RAND-modified Delphi method. A multi-center study was performed and medical charts of 1024 women with a CS and a stratified and weighted randomly selected group of 1036 women with a vaginal delivery were analysed. Quality indicator frequency and adherence were scored in 2060 women with a CS or vaginal delivery. Results The expert panel developed 16 indicators on planned CS and 11 indicators on unplanned CS. Indicator adherence was calculated, defined as the number of women in a specific obstetrical situation in which care was performed as recommended in both planned and unplanned CS settings. The most frequently occurring obstetrical situations with low indicator adherence were: 1) suspected fetal distress (frequency 17%, adh