Structural characterisation of outer membrane proteins from Borrelia burgdorferi sensu lato by small-angle X-ray scattering

Forming the interface between the bacterial cell and the host, the outer membrane of Borrelia is known to play a key role in pathogenicity. Although Borrelia burgdorferi sensu lato are considered to be Gram-negative, their outer membrane is unique, lacking liposaccharides and phosphatidylethanolamine. It contains a variety of glycolipids, surface exposed lipoproteins and a number of membrane-spanning β-barrels. BAPKO_0422, BB_0562 and BG_0408 are membrane proteins, theoretically predicted to form 8-stranded, membrane-spanning β-barrels. The aim of this work is to produce recombinant versions of these proteins, and determine molecular envelopes by small-angle X-ray scattering (SAXS). The β-barrel model can then be tested by comparing the experimental molecular envelope with the theoretical predictions. Three Borrelia proteins (BAPKO_0422, BB_0562 and BG_0408) were recombinantly expressed in the E. coli expression system using the pET-47 expression vector. The putative membrane proteins were purified by immobilised metal affinity chromatography (IMAC). The His tag of BAPKO_0422 was enzymatically removed to produce a native protein, and to allow for a visual comparison of the protein both with and without the His tag. SAXS data for each protein were collected and the overall shape was determined using ab initio methods. The pair-distance distribution function (P(r) function) of BAPKO_0422 with the His tag indicated a particle overlap potentially caused by the flexible 6-His tag at the N-terminus. Kratky plots of BAPKO_0422, BB_0562 and BG_0408 revealed the parabolic convergence for a folded particle. The low-resolution molecular envelopes of BAPKO_0422, BB_0562 and BG_0408 are consistent with the structure of an 8-stranded β-barrel. The filtered envelopes are in agreement with the shape and size of the E. coli homologue OmpX. The likely orientation of the protein within the outer membrane can be deduced by comparing molecular envelopes with and without the N-terminal His tag. The data suggest that BAPKO_0422, BB_0562 and BG_0408 are single-domain cylindrical-shaped molecules with no evidence of an internal pore. Several questions remain to be answered, such as the oligomeric state of BG_0408 and BAPKO_0422. The function of these 8-stranded β-barrels in the Borrelial outer membrane remains to be investigated

Cotton fabric coated with conducting polymers and its application in monitoring of carnivorous plant response

The paper describes the electrical plant response to mechanical stimulation monitored with the help of conducting polymers deposited on cotton fabric. Cotton fabric was coated with conducting polymers, polyaniline or polypyrrole, in situ during the oxidation of respective monomers in aqueous medium. Thus, modified fabrics were again coated with polypyrrole or polyaniline, respectively, in order to investigate any synergetic effect between both polymers with respect to conductivity and its stability during repeated dry cleaning. The coating was confirmed by infrared spectroscopy. The resulting fabrics have been used as electrodes to collect the electrical response to the stimulation of a Venus flytrap plant. This is a paradigm of the use of conducting polymers in monitoring of plant neurobiology.Web of Science164art. no. 49

Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein

The glycoproteins of hepatitis C virus, E1E2, are unlike any other viral fusion machinery yet described, and are the current focus of immunogen design in HCV vaccine development; thus, making E1E2 both scientifically and medically important. We used pre-existing, but fragmentary, structures to model a complete ectodomain of the major glycoprotein E2 from three strains of HCV. We then performed molecular dynamic simulations to explore the conformational landscape of E2, revealing a number of important features. Despite high sequence divergence, and subtle differences in the models, E2 from different strains behave similarly, possessing a stable core flanked by highly flexible regions, some of which perform essential functions such as receptor binding. Comparison with sequence data suggest that this consistent behaviour is conferred by a network of conserved residues that act as hinge and anchor points throughout E2. The variable regions (HVR-1, HVR-2 and VR-3) exhibit particularly high flexibility, and bioinformatic analysis suggests that HVR-1 is a putative intrinsically disordered protein region. Dynamic cross-correlation analyses demonstrate intramolecular communication and suggest that specific regions, such as HVR-1, can exert influence throughout E2. To support our computational approach we performed small-angle X-ray scattering with purified E2 ectodomain; this data was consistent with our MD experiments, suggesting a compact globular core with peripheral flexible regions. This work captures the dynamic behaviour of E2 and has direct relevance to the interaction of HCV with cell-surface receptors and neutralising antibodies

Bidisperse magnetorheological fluids utilizing composite polypyrrole nanotubes/magnetite nanoparticles and carbonyl iron microspheres

Conductive polypyrrole nanotubes were synthesized with a two-step one-pot synthesis. During synthesis, the nanotubes were decorated with magnetite nanoparticles at different concentrations granting them magnetic properties. The characterization of the tubes revealed differences from the theoretical reactions. A bidisperse magnetorheological fluid (MRF) was prepared by mixing the composite polypyrrole nanotubes/magnetite nanoparticles with commercial carbonyl iron spherical microparticles in silicone oil. The rheological properties of the bidisperse system were studied under the presence of magnetic field at room and elevated temperature. An enhancement of the MR effect with the presence of the nanotubes was observed when compared with a standard MRF consisted only of magnetic microparticles. Due to the faster magnetic saturation of the nanotubes, this enhancement is exceptionally high at low magnetic fields. The stability of the system is studied under dynamic conditions where it is revealed that the nanotubes keep the standard particles well dispersed with the sedimentation improving by more than 50%.Internal Grant Agency of Tomas Bata University in Zlín, (IGA/CPS/2022/004, RP/CPS/2022/003); National Technical Library in Prague; Tomas Bata University in Zlin, TBU; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT, MŠMT; en:MEYS, (RP/CPS/2022/007); Univerzita Tomáše Bati ve ZlíněNational Technical Library in Prague; Internal Grant Agency of Tomas Bata University in Zlin [IGA/CPS/2022/004]; project DKRVO [RP/CPS/2022/007, RP/CPS/2022/003]; Ministry of Education, Youth and Sports of the Czech Republi

Conducting and magnetic hybrid polypyrrole/nickel composites and their application in magnetorheology

Hybrid organic/inorganic conducting and magnetic composites of core–shell type have been prepared by in-situ coating of nickel microparticles with polypyrrole. Three series of syntheses have been made. In the first, pyrrole was oxidised with ammonium peroxydisulfate in water in the presence of various amounts of nickel and the composites contained up to 83 wt% of this metal. The second series used 0.1 M sulfuric acid as a reaction medium. Finally, the composites with polypyrrole nanotubes were prepared in water in the presence of structure-guiding methyl orange dye. The nanotubes have always been accompanied by the globular morphology. FTIR and Raman spectroscopies confirmed the formation of polypyrrole. The resistivity of composite powders of the order of tens to hundreds Ω cm was monitored as a function of pressure up to 10 MPa. The resistivity of composites slightly increased with increasing content of nickel. This apparent paradox is explained by the coating of nickel particles with polypyrrole, which prevents their contact and subsequent generation of metallic conducting pathways. Electrical properties were practically independent of the way of composite preparation or nickel content and were controlled by the polypyrrole phase. On the contrary, magnetic properties were determined exclusively by nickel content. The composites were used as a solid phase to prepare a magnetorheological fluid. The test showed better performance when compared with a different nickel system reported earlier.Grantová Agentura České Republiky, GA ČR, (22-25734S, 23-07244S)Czech Science Foundatio

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human Factor-H and is predicted to form a membrane-spanning beta-barrel

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a Spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The OmpA domain is an eight-stranded membrane-spanning β-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work we describe the identification of four novel OmpA-like β-barrels from Borrelia afzelii, the most common cause of erythema migrans rash in Europe. Structural characterisation of one these proteins (BAPKO_0422) by small angle X-ray scattering (SAXS) and circular dichroism indicate a compact globular structure rich in β-strand consistent with a monomeric β-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25 x 45 Å. Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal Phe residue commonly found in Proteobacterial OM proteins is replaced by Ile/Leu or Asn. BAPKO_0422 is demonstrated to bind human factor-H and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value

An entropic safety catch controls Hepatitis C virus entry and antibody resistance

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery

Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike

The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune-driven antigenic variation and ongoing adaptation to a new host

The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications