An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding.

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference.

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids.

Probing coenzyme A homeostasis with semisynthetic biosensors.

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP-HaloTag fusion protein with a fluorescent ligand.

Sactipeptide Engineering by Probing the Substrate Tolerance of a Thioether-Bond-Forming Sactisynthase.

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus.

Bottom-up assembly of biomedical relevant fully synthetic extracellular vesicles.

Extracellular vesicles (EVs) are fundamental for intercellular communication and influence nearly every process in cell physiology. However, because of their intricate molecular complexity, quantitative knowledge on their signaling mechanisms is missing, particularly impeding their therapeutic application. We used a complementary and quantitative engineering approach based on sequential synthetic bottom-up assembly of fully functional EVs with precisely controlled lipid, protein, and RNA composition.

Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery.

Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biology and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diameters of ~100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.

A biosensor for measuring NAD levels at the point of care.

The cellular level of nicotinamide adenine dinucleotide (NAD), through its different functions, affects cellular metabolism and signalling. A decrease in the NAD content has been associated with various pathologies and physiological aging, while strategies to boost cellular NAD levels have been shown to be effective against age-related diseases in many animal models. The link between decreased NAD levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD, ideally applicable at the point of care.

Semisynthetic sensor proteins enable metabolic assays at the point of care.

Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, light-emitting sensor proteins for use in paper-based metabolic assays.

Differential mobility separation of leukotrienes and protectins.

Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship.

Fragmentation follows structure: top-down mass spectrometry elucidates the topology of engineered cystine-knot miniproteins.

Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity.

Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.

Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals.

Bioconjugation on cube-octameric silsesquioxanes.

Small, compact, and highly symmetric cube-octameric silsesquioxanes have recently attracted increased attention as scaffolds for tailor-made bioconjugates. The expanded arsenal of effective conjugation methods (CuAAC, TEC, oxime ligation) allows one to decorate these nanoparticles bearing up to eight addressable organic substituents, with a wide range of biorelevant ligands, among them carbohydrates, peptides, miniproteins, reporter molecules, and rare-earth chelates. Low toxicity of COSS-based molecules combined with solubility in aqueous systems and half-life sufficient for in vivo studies make these structures attractive targets for a number of applications, among them drug delivery, tumor diagnostics and therapy.

Cube-octameric silsesquioxane-mediated cargo peptide delivery into living cancer cells.

Cube octameric silsesquioxanes (COSS) are among the smallest nanoparticles known to date with a diameter of only 0.7 nm. We describe a COSS-based delivery system which allows for the drug targeting in human cells.

Oxidative folding of peptides with cystine-knot architectures: kinetic studies and optimization of folding conditions.

Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, cystine-knot peptides-commonly named "knottins"-make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide-based pharmaceuticals.

A sensitive method for rapid detection of alkyl halides and dehalogenase activity using a multistep enzyme assay.

A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules.

From pico to nano: biofunctionalization of cube-octameric silsesquioxanes by peptides and miniproteins.

Polyhedral silsesquioxanes are considered valuable conjugation scaffolds. Nevertheless, only a few examples of silsesquioxane-assembled peptide oligomers have been reported to date. We developed a new bioorthogonal cube-octameric silsesquioxane (COSS) scaffold bearing eight aminooxy coupling sites allowing for the conjugation of diverse peptides via oxime ligation.

Towards click bioconjugations on cube-octameric silsesquioxane scaffolds.

Cube-octameric silsesquioxane (POSS) based conjugation scaffolds for copper catalysed azide-alkyne [3+2] cycloaddition are reported. The synthetic route to octaazido and octaalkyno functionalised POSS templates without cage rearrangements is described. A set of click couplings is conducted including the first effective conjugation with a fully unprotected functional peptide towards a POSS assembled peptide octamer.

Application of copper(I) catalyzed azide-alkyne [3+2] cycloaddition to the synthesis of template-assembled multivalent peptide conjugates.

Here we describe the facile generation of tetravalent peptide conjugates via a copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) using a cyclic peptide template as a versatile conjugation scaffold. This stable and rigid framework is a conformationally constrained cyclic beta-sheet decorated with spatially defined alkyne moieties that serve as selectively addressable coupling sites. The proposed method allows for the effective coupling of unprotected peptide monomers in water at room temperature within comparatively short reaction times.