Scientific and practical applications of supported lipid-protein bilayers are described. Membranes can be covalently coupled to or separated from solids by ultrathin layers of water or soft polymer cushions. The latter systems maintain the structural and dynamic properties of free bilayers, forming a class of models of biomembranes that allow the application of a manifold of surface-sensitive techniques. They form versatile models of low-dimensionality complex fluids, which can be used to study interfacial forces and wetting phenomena, and enable the design of phantom cells to explore the interplay of lock-and-key forces (such as receptor-ligand binding) and universal forces for cell adhesion. Practical applications are the design of (highly selective) receptor surfaces of biosensors on electrooptical devices or the biofunctionalization of inorganic solids.

https://science.sciencemag.org/content/sci/271/5245/43.full.pdf

Supported Membranes: Scientific and Practical Applications

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

/pdf/an-updated-evolutionary-classification-of-crispr-cas-systems-3hlvj1kkwk.pdf

An updated evolutionary classification of CRISPR–Cas systems

Isogenic strain construction and gene mapping in Candida albicans.

Universal sample preparation method for proteome analysis

The ATP-binding cassette (ABC) transporters are a family of large proteins in membranes and are able to transport a variety of compounds through membranes against steep concentration gradients at the cost of ATP hydrolysis. The available outline of the human genome contains 48 ABC genes; 16 of these have a known function and 14 are associated with a defined human disease. Major physiological functions of ABC transporters include the transport of lipids, bile salts, toxic compounds, and peptides for antigen presentation or other purposes. We review the functions of mammalian ABC transporters, emphasizing biochemical mechanisms and genetic defects. Our overview illustrates the importance of ABC transporters in human physiology, toxicology, pharmacology, and disease. We focus on three topics: (a) ABC transporters transporting drugs (xenotoxins) and drug conjugates. (b) Mammalian secretory epithelia using ABC transporters to excrete a large number of substances, sometimes against a steep concentration gradient. Several inborn errors in liver metabolism are due to mutations in one of the genes for these pumps; these are discussed. (c) A rapidly increasing number of ABC transporters are found to play a role in lipid transport. Defects in each of these transporters are involved in human inborn or acquired diseases.

/pdf/mammalian-abc-transporters-in-health-and-disease-4l5ns15q3r.pdf

Mammalian ABC Transporters in Health and Disease

Biochemie. Eine Einführung für Mediziner und Naturwissenschaftler. Von Werner Müller‐Esterl.

Lantibiotics are a growing class of antimicrobial peptides, which possess antimicrobial activity against mainly Gram-positive bacteria including the highly resistant strains such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci. In the last decades numerous lantibiotics were discovered in natural habitats or designed with bioengineering tools. In this study, we present an insight in the antimicrobial potential of the natural occurring lantibiotic nisin H from Streptococcus hyointestinalis as well as the variant nisin H F1I. We determined the yield of the heterologously expressed peptide and quantified the cleavage efficiency employing the nisin protease NisP. Furthermore, we analyzed the effect on the modification via mass spectrometry analysis. With standardized growth inhibition assays we benchmarked the activity of pure nisin H and the variant nisin H F1I, and their influence on the activity of the nisin immunity proteins NisI and NisFEG from Lactococcus lactis and the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1. We further checked the antibacterial activity against clinical isolates of Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis via microdilution method. In summary, nisin H and the nisin H F1I variant possessed better antimicrobial potency than the natural nisin A.

/pdf/insights-in-the-antimicrobial-potential-of-the-natural-nisin-ia7csxxah8.pdf

Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H.

Type I secretion systems (T1SS) are ubiquitous transport machineries in Gram-negative bacteria. They comprise a relatively simple assembly of three membrane-localised proteins: an inner-membrane complex composed of an ABC transporter and a membrane fusion protein, and a TolC-like outer membrane component. T1SS transport a wide variety of substrates with broad functional diversity. The ABC transporter hemolysin B (HlyB), for example, is part of the hemolysin A-T1SS in Escherichia coli. In contrast to canonical ABC transporters, an accessory domain, a C39 peptidase-like domain (CLD), is located at the N-terminus of HlyB and is essential for secretion. In this study, we have established an optimised purification protocol for HlyB and the subsequent reconstitution employing the saposin-nanoparticle system. We point out the negative influence of free detergent on the basal ATPase activity of HlyB, studied the influence of a lysolipid or lipid matrix on activity and present functional studies with the full-length substrate proHlyA in its folded and unfolded states, which both have a stimulatory effect on the ATPase activity.

/pdf/functional-reconstitution-of-hlyb-a-type-i-secretion-abc-1xh81zienc.pdf

Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles.

The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties

SUMMARY Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus (Linne, 1758), catalyses the NADH-dependent condensation of l-arginine and pyruvate to d-octopine, NAD + and water during escape swimming and subsequent recovery. During exercise, ATP is mainly provided by the transphosphorylation of phospho-l-arginine and to some extent by anaerobic glycolysis. NADH resulting from the glycolytic oxidation of 3-phosphoglyceraldehyde to 1,3-bisphosphoglycerate is reoxidized during d-octopine formation. In some scallops d-octopine starts to accumulate during prolonged, strong muscular work, whereas in other species d-octopine formation commences towards the end of swimming and continues to rise during subsequent recovery. The activity of OcDH is regulated by a mandatory, consecutive mode of substrate binding in the order NADH, l-arginine and pyruvate, as demonstrated by isothermal titration calorimetry. The first regulatory step in the forward reaction comprises the binding of NADH to OcDH with a dissociation constant K d of 0.014±0.006 mmol l –1 , which reflects a high affinity and tight association of the apoenzyme with the co-substrate. In the reverse direction, NAD + binds first with a K d of 0.20±0.004 mmol l –1 followed by d-octopine. The binary OcDH–NADH complex associates with l-arginine with a K d of 5.5±0.05 mmol l –1 . Only this ternary complex combines with pyruvate, with an estimated K d of approximately 0.8 mmol l –1 as deduced from pyruvate concentrations determined in the muscle of exhausted scallops. At tissue concentrations of pyruvate between 0.5 and 1.2 mmol l –1 in the valve adductor muscle of fatigued P. maximus , binding of pyruvate to OcDH plays the most decisive role in initiating OcDH activity and, therefore, in controlling the onset of d-octopine formation.

Lutz Schmitt

Papers

Biochemie. Eine Einführung für Mediziner und Naturwissenschaftler. Von Werner Müller‐Esterl.

Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H.

Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles.

The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties

Control of d-octopine formation in scallop adductor muscle as revealed through thermodynamic studies of octopine dehydrogenase