1,720,992 research outputs found
Structural studies of a membrane embedded chloride channel from E.Coli, a prokaryotic homologue of ClC channels
Full text linkThe ClC family of chloride channels are found throughout the eukaryotes and homologues are found among the prokaryotes. There are nine ClC chloride channels found in mammals that are expressed either ubiquitously or in specific tissues. They have a role in a broad spectrum of cellular functions, ranging from the control of electrical excitability of neurons and the maintenance of the balance of charge required in the acidification of organelles, to the regulation of cell volume. The physiological importance of these channels is often demonstrated by disease causing mutations. Mutations in ClC chloride channels cause myotonias, Becker’s Disease and have been implicated in the spread of cancer. The structural information on members of the ClC family is extremely limited. For this reason ClC prokaryotic homologues, ecClC1 and ecClC2 from E.coli, were cloned and expressed in the hope that this system would produce sufficient amounts of protein to grow crystals in order to obtain three-dimensional information. This information will provide a detailed mechanism of chloride conduction and molecular architecture of the channel necessary for the further physiological and pharmacological characterisation of this important family of chloride channels. The E.coli ClC chloride channel ecClC was cloned from E.coli genomic DNA and overexpressed in E.coli. The recombinant protein was purified from the E.coli membranes using detergents. Metal affinity and size exclusion chromatography were used to purify the protein to near homogeneity. Crystals of ecClC2 have been grown that diffract to 4Å and structural analysis is underway. This thesis describes the development of the purification protocol, the crystallisation of the protein and the preliminary analysis of the X-ray diffraction data
The N-acyltransferase Lnt: Structure-function insights from recent simultaneous studies
Full text linkBacterial lipoproteins have been researched for decades due to their roles in a large number of biological functions. There were no structures of their main three membrane processing enzymes, until 2016 for Lgt and LspA, and then 2017 for Lnt with not one but three simultaneous, independent publications. We have analyzed the recent findings for this apolipoprotein N-acyltransferase Lnt, with comparisons between the novel structures, and with soluble nitrilases, to determine the significance of unique features in terms of substrate's recognition and binding mechanism influenced by exclusive residues, two transmembrane helices, and a flexible loop
Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms
No full textAbstract: PDZ domains most commonly bind the C-terminus of their protein targets. Typically theC-terminal four residues of the protein target are considered as the binding motif, particularly theC-terminal residue (P0) and third-last residue (P-2) that form the major contacts with the PDZdomain’s ‘‘binding groove’’. We solved crystal structures of seven human PDZ domains, includingfive of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7show a binding mode with only the C-terminal P0 residue bound in the binding groove.Importantly, in some cases, the P-2 residue formed interactions outside of the binding groove,providing insight into the influence of residues remote from the binding groove on selectivity. Inthe GRASP structure, we observed both canonical and noncanonical binding in the two moleculespresent in the asymmetric unit making a direct comparison of these binding modes possible. Inaddition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allowcomparison with canonical binding for the PDLIM PDZ domain family. Although influenced bycrystal packing arrangements, the structures nevertheless show that changes in the positions ofPDZ domain side-chains and the aB helix allow noncanonical binding interactions. Theseinteractions may be indicative of intermediate states between unbound and fully bound PDZdomain and target protein. The noncanonical ‘‘perpendicular" binding observed potentiallyrepresents the general form of a kinetic intermediate. Comparison with canonical binding suggeststhat the rearrangement during binding involves both the PDZ domain and its ligand.Keywords: PDZ domain; X-ray structure; binding mode; substrate selectivity; binding mechanis
Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance
No full textBackground: the E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts.Results: using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins.Conclusions: we demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.</p
Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae
No full textThe inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels
Concentration-dependent effects of acute and chronic neonicotinoid exposure on the behaviour and development of the nematode Caenorhabditis elegans
No full textBackground: neonicotinoids insecticides are under review due to emerging toxicity to non-target species. Interest has focused on biological pollinators whilst their effects on other organisms that are key contributors to the ecosystem remain largely unknown. To advance this we have tested the effects of representatives of three major classes of neonicotinoids, thiacloprid, clothianidin and nitenpyram on the free-living nematode Caenorhabditis elegans (C. elegans), as a representative of the Nematoda, an ecologically important phylum contributing to biomass. Results: concentrations that are several-fold higher than those with effects against target species had limited impact on locomotor function. However, increased potency was observed in a mutant with a hyper-permeable cuticle which shows that drug access limits the effects of the neonicotinoids in C. elegans. Thiacloprid was most potent (EC50 714 µM). In addition, it selectively delayed larval development in wild-type worms at 1 mM. Conclusions: C. elegans is less susceptible to neonicotinoids than target species of pest insect. We discuss an approach in which this defined low sensitivity may be exploited by heterologous expression of insect nicotinic acetylcholine receptors from both pest and beneficial insects, in transgenic C. elegans with increased cuticle permeability to provide a whole organism assay for species-dependent neonicotinoid effects. <br/
Direct and specific activation of human inward rectifier K+ channels by membrane phosphatidylinositol 4,5-bisphosphate
No full textMany ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)), but studies examining the PIP(2) dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP(2) dependence of purified human Kir2.1 and Kir2.2 activity using (86)Rb(+) flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP(2) and that PIP(2) is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP(2) levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP(2), and open probability increases to ?0.4 at 1% PIP(2). Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP(2); PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P(3) causes minimal activity. The PIP(2) dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP(2). This raises the interesting hypothesis that PIP(2) activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP(2) in the eukaryotic cell membrane
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Dual-mode phospholipid regulation of human inward rectifying potassium channels
No full textThe lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity
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