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Formation of unilamellae of fully hydrated dipalmitoylphosphatidylcholine (DPPC) was induced by a horn sonicator from multilamellar vesicles and followed by time-resolved synchrotron small angle X-ray scattering and direct visual morphological investigations by the means of transmission electron-microscopy combined with freeze-fracture. Without incubation the ultrasonication causes continuous increasing in temperature and transformation from the gel to rippled gel structures, then reaching the main transition, the formfactor of unilamellar structure appeared. The ultrasonication resulted in different layer formations at the characteristic temperatures of the gel (20 °C), rippled gel (38 °C), and liquid crystalline (45 °C) phases of the system. At 20 °C irregular stacks of multi and oligolamellar lamellae were shaped even after three hours of ultrasonication. At 38 and 45 °C the ultrasound induced dominantly unilamellar vesicles (ULVs) in a short time (10 and 3 – 5 min, respectively, under typical ultrasound treatments in the general laboratory practice). After the end of the ultrasonication, irregular layer formations with defects structures increased when the temperature of sonication was above the chain melting temperature of the hydrated DPPC system, underlining the importance of optimized sonication processes.

Abstract Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral β3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.

Non-iridescent structural plumage reflectance is a sexually selected indicator of individual quality in several bird species. However, the structural basis of individual differences remains unclear. In particular, the dominant periodicity of the quasi-ordered feather barb nanostructure is of key importance in colour generation, but no study has successfully traced back reflectance parameters, and particularly hue, to nanostructural periodicity, although this would be key to deciphering the information content of individual variation. We used matrix small-angle X-ray scattering measurements of intact, stacked feather samples from the blue tit crown to estimate the sex-dependence and individual variation of nanostructure and its effects on light reflectance. Measures of nanostructural periodicity successfully predicted brightness, ultraviolet chroma and also hue, with statistically similar effects in the two sexes. However, we also observed a lack of overall effect of the nanostructural inhomogeneity estimate on reflectance chromaticity, sex-dependent accuracy in hue prediction and strong sex-dependence in position estimation error. We suggest that reflectance attributes are modified by other feather structures in a sex-specific manner, and that within-individual variation in nanostructural parameters exists within or among feathers and this confounds the interpretation of structure–reflectance relationships at the plumage area level.

β-peptides are peptide-mimetics with wide biomedical and biotechnological applications. Their most peculiar feature is their readiness to fold into various secondary structures not found in their natural counterparts. Furthermore, β-amino acid insertions to natural polypeptides were found to provide unique features and also increase enzymatic stability. This is due to the additional methylene group in their backbone, which on the other hand makes it impossible to use the established algorithms and computational tools developed for α-peptides and proteins. An extension to the PyMOL v2.x molecular graphics environment is presented for β-peptides, which allows rapid graphical visualization and greatly simplified model building for computational studies, with special emphasis also put on direct formation of diverse folding states.

The red-emitting fluorescent properties of bovine serum albumin (BSA)–gold conjugates are commonly attributed to gold nanoclusters formed by metallic and ionized gold atoms, stabilized by the protein. Others argue that red fluorescence originates from gold cation–protein complexes instead, not gold nanoclusters. Our fluorescence and infrared spectroscopy, neutron, and X-ray small-angle scattering measurements show that the fluorescence and structural behavior of BSA–Au conjugates are different in normal and heavy water, strengthening the argument for the existence of loose ionic gold–protein complexes. The quantum yield for red-emitting luminescence is higher in heavy water (3.5%) than normal water (2.4%), emphasizing the impact of hydration effects. Changes in red luminescence are associated with the perturbations of BSA conformations and alterations to interatomic gold–sulfur and gold–oxygen interactions. The relative alignment of domains I and II, II and III, III and IV of BSA, determined from small-angle scattering measurements, indicate a loose (“expanded-like”) structure at pH 12 (pD ~12); by contrast, at pH 7 (pD ~7), a more regular formation appears with an increased distance between the I and II domains, suggesting the localization of gold atoms in these regions.

Staphylococcus aureus causes localized infections or invasive diseases (abscesses or endocarditis). One of its virulence factors is staphylocoagulase (SCG), which binds prothrombin to form a complex with thrombin-like proteolytic activity and leads to uncontrolled fibrin generation at sites of bacterial inoculation. The aim of this study was to characterize the formation, structure, mechanical properties and lysis of SCG-generated clots. Recombinant SCG was expressed in Escherichia coli, purified and the amidolytic activity of its complexes with human prothrombin (SCG-PT) and thrombin (SCG-T) was determined using human thrombin as a reference. Fibrin clots were prepared from purified fibrinogen and human plasma using thrombin, SCG-PT or SCG-T as a coagulase. The kinetics of clot formation and lysis by tissue-type plasminogen activator (tPA) were monitored with turbidimetric assays. Fibrin ultrastructure was examined with scanning electron microscopy and small-angle X-ray scattering (SAXS). Fibrin clot porosity was characterized with fluid permeation assays, whereas the viscoelastic properties and mechanical stability were evaluated with oscillation rheometry. Compared to thrombin, the amidolytic and clotting activity of SCG-PT was 1.6- to 2.5-fold lower on a molar basis. SCG-T had equivalent amidolytic, but reduced clotting activity both on pure fibrinogen (1.6-fold), and in plasma (1.3-fold). The SCG-PT and SCG-T generated fibrin with thicker fibers (10–60% increase in median diameter) than thrombin due to increased number of fibrin protofibrils per fiber cross-section. According to the fluid permeability of the clots SCG-PT and SCG-T promoted the formation of more porous structures. The shear stress resistance in the pure fibrin and plasma clots generated by SCG-PT was significantly lower than in the thrombin clots (243.8 ± 22.0 Pa shear stress was sufficient for disassembly of SCG-PT fibrin vs. 937.3 ± 65.6 Pa in thrombin clots). The tPA-mediated lysis of both pure fibrin and plasma clots produced by SCG-PT or SCG-T was accelerated compared to thrombin, resulting in up to a 2.1-fold increase in tPA potency. Our results indicate that SCG generates a thrombus scaffold with a structure characterized by impaired mechanical stability and increased lytic susceptibility. This proneness to clot disintegration could have implications in the septic embolism from endocardial bacterial vegetation.