Structural studies suggest aggregation as one of the modes of action for teixobactin

Teixobactin is a new promising antibiotic that targets cell wall biosynthesis by binding to lipid II and has no detectable resistance thanks to its unique but yet not fully understood mechanism of operation. To aid in the structure-based design of teixobactin analogues with improved pharmacological properties, we present a 3D structure of native teixobactin in membrane mimetics and characterise its binding to lipid II through a combination of solution NMR and fast (90 kHz) magic angle spinning solid state NMR. In NMR titrations, we observe a pattern strongly suggesting interactions between the backbone of the C-terminal “cage” and the pyrophosphate moiety in lipid II. We find that the N-terminal part of teixobactin does not only act as a membrane anchor, as previously thought, but is actively involved in binding. Moreover, teixobactin forms a well-structured and specific complex with lipid II, where the N-terminal part of teixobactin assumes a b conformation that is highly prone to aggregation, which likely contributes to the antibiotic's high bactericidal efficiency. Overall, our study provides several new clues to teixobactin's modes of action

Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules

Investigating interactions using solid state NMR: applications to biomolecular complexes

Solid state NMR is a powerful method to obtain information on structure and dynamics of proteins, protein complexes, and other biomolecular assemblies, that due to solubility and size limitations cannot be achieved by other methods. This thesis is dedicated to the use of proton detected solid state NMR experiments at fast (60-100 kHz) magic angle spinning speeds to probe biomolecular structure, dynamics and intermolecular interactions. We used solvent paramagnetic relaxation enhancements to (i) speed up experiments and (ii) measure solvent accesibililty to map protein – protein interfaces. Further we use nuclear spin relaxation measurements to obtain information on protein dynamics in a small protein in two different ensembles; crystalline and precipitated in a > 300 kDa complex. We also apply a combined solution and solid state NMR approach to investigate the interactions between possibly the most promising antibiotic lead in modern time, teixobactin, and arguably the most exciting bacterial target, lipid II. Our most important results include a new method for probing protein-protein interactions using solvent paramagnetic relaxation enhancements, the first site specific dynamics measurements spanning a wide range of time scales obtained in a large protein complex using as little as 8 nanomoles of isotopically labelled material, the use of solvent paramagnetic relaxation enhancements to enable measurements of conformational exchange in a large protein complex and finally we have identified important conformational changes involved in the binding of teixobactin to cell wall precursor lipid II

Konsumenters attityd gentemot etisk produktion: En studie om ägg : En undersökning om hur etisk produktion påverkar konsumenters attityd och betalningsvillighet

Syftet med denna studie är att undersöka hur etisk produktion påverkar konsumenters attityd gentemot en ökad investering vid konsumtion. Vedertagen teori menar på att etisk produktion har en positiv påverkan på den genomsnittlige konsumentens attityd och att konsumenter kan tänka sig att betala ett högre pris för produkter producerade på etiskt vis. Med syfte att konkretisera begreppet CSR och underlätta för respondenterna i enkätundersökningen används begreppet etisk produktion med hänvisning till äggkonsumtion. Aspekterna av begreppet etisk produktion som ligger till grund för denna studie är miljövänlig transport och god hönshållning. Studiens resultat redogör för att konsumenter är positivt inställda till att betala ett högre pris för produkter producerade på ett etiskt vis och att etisk produktion är ett produktattribut som påverkar konsumenternas attityd gentemot konsumtion

Konsumenters attityd gentemot etisk produktion: En studie om ägg : En undersökning om hur etisk produktion påverkar konsumenters attityd och betalningsvillighet

Syftet med denna studie är att undersöka hur etisk produktion påverkar konsumenters attityd gentemot en ökad investering vid konsumtion. Vedertagen teori menar på att etisk produktion har en positiv påverkan på den genomsnittlige konsumentens attityd och att konsumenter kan tänka sig att betala ett högre pris för produkter producerade på etiskt vis. Med syfte att konkretisera begreppet CSR och underlätta för respondenterna i enkätundersökningen används begreppet etisk produktion med hänvisning till äggkonsumtion. Aspekterna av begreppet etisk produktion som ligger till grund för denna studie är miljövänlig transport och god hönshållning. Studiens resultat redogör för att konsumenter är positivt inställda till att betala ett högre pris för produkter producerade på ett etiskt vis och att etisk produktion är ett produktattribut som påverkar konsumenternas attityd gentemot konsumtion

Konsumenters attityd gentemot etisk produktion: En studie om ägg : En undersökning om hur etisk produktion påverkar konsumenters attityd och betalningsvillighet

Syftet med denna studie är att undersöka hur etisk produktion påverkar konsumenters attityd gentemot en ökad investering vid konsumtion. Vedertagen teori menar på att etisk produktion har en positiv påverkan på den genomsnittlige konsumentens attityd och att konsumenter kan tänka sig att betala ett högre pris för produkter producerade på etiskt vis. Med syfte att konkretisera begreppet CSR och underlätta för respondenterna i enkätundersökningen används begreppet etisk produktion med hänvisning till äggkonsumtion. Aspekterna av begreppet etisk produktion som ligger till grund för denna studie är miljövänlig transport och god hönshållning. Studiens resultat redogör för att konsumenter är positivt inställda till att betala ett högre pris för produkter producerade på ett etiskt vis och att etisk produktion är ett produktattribut som påverkar konsumenternas attityd gentemot konsumtion

Åtkomststudie för robotiserad svetsning av flygmotordetalj

The aim of this thesis was to investigate if the robotized welding method FSW (Friction Stir Welding) could be applied for joining a rotating structure in an aero engine at Volvo Aero Corporation. FSW is expected to introduce less defects than today’s welding methods and could therefore be suitable for critical aero components. The material is the nickel based alloy Inconel 718, however a material experimentation is outside the scope of this report.The main goal of this study is to verify if the ESAB ROSIO robot based FSW-system has a suitable work space to be able to weld the rotating structure, and if the welding tool has accessibility to the joints. The FSW-process needs a rigid fixture, and a number of fix-ture concepts are presented based on a proposed weld sequence. A final fixture design is proposed, which requires a new design of the structure.The accessibility studies were performed in the robot simulations software Robot Stu-dio. This showed that the robot was unable to weld the proposed model in all areas in the original design. If the robot and the rotating structure can be redesigned the access will be achieved as shown in Robot Studio simulations.In order to be implemented in real production a number of further actions need to be taken and the result of this study can be a basis for these

Structural Plasticity of the Selectivity Filter in Cation Channels

Ion channels allow for the passage of ions across biological membranes, which is essential for the functioning of a cell. In pore loop channels the selectivity filter (SF) is a conserved sequence that forms a constriction with multiple ion binding sites. It is becoming increasingly clear that there are several conformations and dynamic states of the SF in cation channels. Here we outline specific modes of structural plasticity observed in the SFs of various pore loop channels: disorder, asymmetry, and collapse. We summarize the multiple atomic structures with varying SF conformations as well as asymmetric and more dynamic states that were discovered recently using structural biology, spectroscopic, and computational methods. Overall, we discuss here that structural plasticity within the SF is a key molecular determinant of ion channel gating behavior.Peer Reviewe

ACG126/8f: Aprobación de la Carta de Servicios Sociales al Personal de la Universidad de Granada

Solid state nMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate 15n R1ρ relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a >300 kDa precipitated complex of GB1 with full length human immunoglobulin G (igG). the addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 μg of labelled material in 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the β-sheet in GB1 in crystals and in complex with IgG. However, the slow motion observed for a number of residues in the α-helix of crystalline GB1 is not detected in the complex

Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-Silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle x-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution