203 research outputs found
Investigation of insect olfactory receptor modulation
No full textP4-ATPases have recently emerged as a target for structural studies due to their roles in cellular function. These enzymes actively transport phospholipids from the exoplasmic leaflet to the cytosolic leaflet of eukaryotic membranes, establishing and regulating lipid composition asymmetry between the two leaflets of the membrane bilayer. This asymmetry plays a crucial role in various cellular processes, including vesicle budding, cell signalling, and apoptosis, among others. In Drosophila melanogaster, olfactory receptors play a vital role in odorant perception and are modulated by several olfactory sensory neuron (OSN) proteins and non-OSN proteins. Intriguingly, recent studies have implicated flippases as crucial players in olfactory receptor expression and odorant sensitivity regulation in Drosophila melanogaster. P4-ATPases, being large membrane proteins forming heteromeric complexes, present several challenges for in-vitro studies and structural determination. Here, we investigate the structural and biochemical characterisation of the P4-ATPase dATP8B, a flippase in Drosophila, and its interaction with the chaperone protein dCDC50A. Initial analysis revealed large unstructured N- and C-terminal regions in dATP8B, hypothesized to contribute to instability and purification challenges. To address this, new constructs were designed with targeted deletions of these unstructured regions. Our findings demonstrate that flippase and dCDC50A in Drosophila melanogaster forms a tight and stable complex.Bachelor's degre
Structures and regulation of coupling subunit F and the arrangement of the subunit DF-assembly in the Saccharomyces cerevisiae v1vO ATPase
No full textV-ATPases play an important role in the acidification of intracellular compartments such as lysosomes, endosomes, Golgi complexes and secretary granules. The V-ATPases are composed of at least 14 separate gene products, with many of these subunits present in multiple isoforms. The proposed subunit stoichiometry of V1 is A3:B3:C:D:E3:F:G3:H1 (1). The integral VO domain contains six different subunits in a stoichiometry of a:d:c4-5:c’:c”:e. V-ATPases exist in a dynamic equilibrium between fully assembled complexes and reversibly disassembled V1 and VO subcomplexes. Depending on the energy status of the cell, this equilibrium can be rapidly shifted (2). Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A3B3 sector of the V1 ATPase to pump protons via the membrane-embedded VO sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F belongs. In the present study, the low resolution structure of recombinant subunit F (VMA7p) of the eukaryotic V-ATPase from Saccharomyces cerevisiae has been analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5 nm long egglike shaped region, connected via a 1.5 nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% -helix, 32% -sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F1-94 was produced and analyzed by SAXS. Comparison of the F1-94 shape with the shape of the entire subunit F showed the missing hook-like region in F1-94, supported by the decreased Dmax value of F1-94 and indicating that the hook-like region consists of the C-terminal residues (3). The NMR solution structure of the C-terminal peptide, F90-116, was solved, showing an α-helical region between residues 103-113 (3). The F90-116 solution structure fitted well in the hook-like region of subunit F (3). In order to understand the structural features of F1-94 at the atomic level, X-ray crystallography was performed. The crystal structure of F1-94 reveals a Rossmann fold with alternating β-strands and α-helices (4). Elliptical shaped F1-94 has four β-strands which are surrounded by four α-helices. F1-94 contains two important loops spanning between α1-β2 (26GQITPETQEK35) and α2-β3 (60ERDDI64) which are present only in eukaryotic F subunits. Multiple sequence alignments of subunit F show that the 60ERDDI64 loop is highly conserved among the eukaryotic V-ATPases (4). NMR spectroscopy of the entire subunit F confirmed the secondary structural features of the crystallographic structure F1-94 in solution as well as the C-terminal peptide, F90-116. The heteronuclear NOE experiment shows that subunit F has a rigid core in the N-terminal domain, whereas α1 and α5 are more flexible in the solution (4). To understand the cross-talk between central stalk subunits with the neighboring subunits, a DF-heterodimer was generated. The DF-heterodimer binds to subunit d with a dissociation constant (Kd) of 52.9 µM as determined by ITC experiment (4). The DF-heterodimer yielded crystals with a dimension of 0.13 mm x 0.10 mm x 0.04 mm, which diffracted maximum to 5 Å.DOCTOR OF PHILOSOPHY (SBS
A multiphase phase-field study of three-dimensional martensitic twinned microstructures at large strains
No full textA thermodynamically consistent multiphase phase-field approach for stress and temperature-induced martensitic phase transformation at the nanoscale and under large strains is developed. A total of N independent order parameters are considered for materials with N variants, where one of the order parameters describes A M transformations and the remaining N-1 independent order parameters describe the transformations between the variants. A non-contradictory gradient energy is used within the free energy of the system to account for the energies of the interfaces. In addition, a non-contradictory kinetic relationships for the rate of the order parameters versus thermodynamic driving forces is suggested. As a result, a system of consistent coupled Ginzburg-Landau equations for the order parameters are derived. The crystallographic solution for twins within twins is presented for the cubic to tetragonal transformations. A 3D complex twins within twins microstructure is simulated using the developed phase-field approach and a large-strain-based nonlinear finite element method. A comparative study between the crystallographic solution and the simulation result is presented.This is a pre-print of the article Basak, Anup, and Valery I. Levitas. "A multiphase phase-field study of three-dimensional martensitic twinned microstructures at large strains." arXiv preprint arXiv:2206.12576 (2022).
DOI: 10.48550/arXiv.2206.12576.
Copyright 2022 The Author(s).
Attribution 4.0 International (CC BY 4.0).
Posted with permission
Failure mechanisms in lithium silicon batteries
No full textLithium silicon (Li-Si) batteries offer more than ten times the theoretical specific capacity compared to current lithium ion battery technologies, by using a silicon anode. In practice however, the cycle life of Li-Si batteries is very limited. The large volume change of the silicon anode is known to be the main reason for this. Research on the volume changes during varying cell cycles and voltages is presented in this thesis and an experimental set up for a quasi in situ study of the SEI layer is suggested. Cycling tests with an amorphous silicon thin film of 220 nm deposited using magnetron sputtering on a copper foil current collector confirmed that the major cause of capacity loss is swelling of the silicon during lithiation, causing the silicon to detach from the current collector and resulting in significant capacity loss. Increasing the lower cut off voltage from 0 V to 0.2 V resulted in a slight improvement of cycle life. Silicon detachment also decreased as determined by SEM images. EFTEM and EDX mapping showed a clear split between a partially lithiated silicon layer on the surface and a pure silicon layer on the current collector side. It can be concluded that discharging Li-Si batteries to 0.2 V instead of 0 V is a promising method to reduce the swelling of silicon during lithiation.HREMQuantum NanoscienceApplied Science
Expression and purification strategies for nanobodies and their interaction studies with Drosophila olfactory co-receptor (Orco) for structural analysis
No full textThis study aimed to stabilise the Olfactory co-receptor of Drosophila melanogaster (Dorco) using a
nanobody-based structural stabilization approach to provide insights into its structure
and function.Bachelor's degre
The cloning expression, purification, and reconstitution of Olfactory receptor co-receptor (Orco) in nanodisc from insects for structural and function studies
No full textInsect vectors host-seeking behaviours are being mediated by the olfactory system, making it an attractive target for managing vector-borne disease transmission. Odorant receptors (ORs) allow insects to recognize a large variety of odorant molecules based on simple combinatorial signalling. The normal function of ORs in insects is regulated by the highly conserved Orco protein, and a diverse tuning subunit. We can study a simplified Orco homotetramer to better understand the structure and function of ORs. Here, we investigated Drosophila melanogaster and Anopheles gambiae Orco tetramers. We have introduced a thermostabilized fusion protein apocytochrome b562RIL (BRIL) domain fused in the intracellular loop of Orco proteins to enable binding of BRIL antibody to protein, which further aids in structure determination by changing properties of Orco receptors. All recombinant Orco-BRIL constructs were successfully cloned Bacmid vectors, for transfection into Sf9 cells (Spodoptera frugiperda) for baculovirus generation, which was confirmed by agarose gel electrophoresis, sequencing, and PCR. The recombinant Orco-BRIL protein was effectively overexpressed in Sf9 cells and purified and reconstituted into a lipid nanodisc, determined by SDS-PAGE, western blot, negative staining electron microscopy. These findings suggests that recombinant Orco-BRIL homotetramer can be efficiently expressed and purified for downstream cryo-EM structural studies.Bachelor of Science in Biological Science
Author response: Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel
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