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Crystallization of membrane protein

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https://www.readbyqxmd.com/read/28089451/crystallogenesis-of-membrane-proteins-mediated-by-polymer-bounded-lipid-nanodiscs
#1
Jana Broecker, Bryan T Eger, Oliver P Ernst
For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene-maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization...
January 3, 2017: Structure
https://www.readbyqxmd.com/read/28077872/structural-basis-for-nutrient-acquisition-by-dominant-members-of-the-human-gut-microbiota
#2
Amy J Glenwright, Karunakar R Pothula, Satya P Bhamidimarri, Dror S Chorev, Arnaud Baslé, Susan J Firbank, Hongjun Zheng, Carol V Robinson, Mathias Winterhalter, Ulrich Kleinekathöfer, David N Bolam, Bert van den Berg
The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG...
January 11, 2017: Nature
https://www.readbyqxmd.com/read/28076802/peldor-spectroscopy-reveals-two-defined-states-of-a-sialic-acid-trap-transporter-sbp-in-solution
#3
Janin Glaenzer, Martin F Peter, Gavin H Thomas, Gregor Hagelueken
The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP...
January 10, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28073257/protein-containing-lipid-bilayers-intercalated-with-size-matched-mesoporous-silica-thin-films
#4
Simon Isaksson, Erik B Watkins, Kathryn L Browning, Tania Kjellerup Lind, Marité Cárdenas, Kristina Hedfalk, Fredrik Höök, Martin Andersson
Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein-material interactions and a lack of physical space...
January 11, 2017: Nano Letters
https://www.readbyqxmd.com/read/28069938/crystal-structure-and-mechanistic-basis-of-a-functional-homolog-of-the-antigen-transporter-tap
#5
Anne Nöll, Christoph Thomas, Valentina Herbring, Tina Zollmann, Katja Barth, Ahmad Reza Mehdipour, Thomas M Tomasiak, Stefan Brüchert, Benesh Joseph, Rupert Abele, Vincent Oliéric, Meitian Wang, Kay Diederichs, Gerhard Hummer, Robert M Stroud, Klaas M Pos, Robert Tampé
ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells...
January 9, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28067914/structure-of-the-immature-zika-virus-at-9-%C3%A3-resolution
#6
Vidya Mangala Prasad, Andrew S Miller, Thomas Klose, Devika Sirohi, Geeta Buda, Wen Jiang, Richard J Kuhn, Michael G Rossmann
The current Zika virus (ZIKV) epidemic is characterized by severe pathogenicity in both children and adults. Sequence changes in ZIKV since its first isolation are apparent when pre-epidemic strains are compared with those causing the current epidemic. However, the residues that are responsible for ZIKV pathogenicity are largely unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of the immature ZIKV at 9-Å resolution. The cryo-EM map was fitted with the crystal structures of the precursor membrane and envelope glycoproteins and was shown to be similar to the structures of other known immature flaviviruses...
January 9, 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28067269/proteomic-analysis-of-cry2aa-binding-proteins-and-their-receptor-function-in-spodoptera-exigua
#7
Lin Qiu, Boyao Zhang, Lang Liu, Weihua Ma, Xiaoping Wang, Chaoliang Lei, Lizhen Chen
The bacterium Bacillus thuringiensis produces Crystal (Cry) proteins that are toxic to a diverse range of insects. Transgenic crops that produce Bt Cry proteins are grown worldwide because of their improved resistance to insect pests. Although Bt "pyramid" cotton that produces both Cry1A and Cry2A is predicted to be more resistant to several lepidopteran pests, including Spodoptera exigua, than plants that produce Cry1Ac alone, the mechanisms responsible for the toxicity of Cry2Aa in S. exigua are not well understood...
January 9, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28065508/kobuviral-non-structural-3a-proteins-act-as-molecular-harnesses-to-hijack-the-host-acbd3-protein
#8
Martin Klima, Dominika Chalupska, Bartosz Różycki, Jana Humpolickova, Lenka Rezabkova, Jan Silhan, Adriana Baumlova, Anna Dubankova, Evzen Boura
Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein...
December 21, 2016: Structure
https://www.readbyqxmd.com/read/28065269/methods-for-structural-and-functional-analyses-of-intramembrane-prenyltransferases-in-the-ubia-superfamily
#9
Y Yang, N Ke, S Liu, W Li
The UbiA superfamily is a group of intramembrane prenyltransferases that generate lipophilic compounds essential in biological membranes. These compounds, which include various quinones, hemes, chlorophylls, and vitamin E, participate in electron transport and function as antioxidants, as well as acting as structural lipids of microbial cell walls and membranes. Prenyltransferases producing these compounds are involved in important physiological processes and human diseases. These UbiA superfamily members differ significantly in their enzymatic activities and substrate selectivities...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28056928/new-ligation-independent-cloning-vectors-for-expression-of-recombinant-proteins-with-a-self-cleaving-cpd-6xhis-tag
#10
Marco Biancucci, Jazel S Dolores, Jennifer Wong, Sarah Grimshaw, Wayne F Anderson, Karla J F Satchell, Keehwan Kwon
BACKGROUND: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. RESULTS: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag"...
January 5, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28052259/conformational-freedom-of-the-lrp6-ectodomain-is-regulated-by-n-glycosylation-and-the-binding-of-the-wnt-antagonist-dkk1
#11
Kyoko Matoba, Emiko Mihara, Keiko Tamura-Kawakami, Naoyuki Miyazaki, Shintaro Maeda, Hidenori Hirai, Samuel Thompson, Kenji Iwasaki, Junichi Takagi
LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules...
January 3, 2017: Cell Reports
https://www.readbyqxmd.com/read/28045456/redox-potential-as-a-master-variable-controlling-pathways-of-metal-reduction-by-geobacter-sulfurreducens
#12
Caleb E Levar, Colleen L Hoffman, Aubrey J Dunshee, Brandy M Toner, Daniel R Bond
Geobacter sulfurreducens uses at least two different pathways to transport electrons out of the inner membrane quinone pool before reducing acceptors beyond the outer membrane. When growing on electrodes poised at oxidizing potentials, the CbcL-dependent pathway operates at or below redox potentials of -0.10 V vs the standard hydrogen electrode, whereas the ImcH-dependent pathway operates only above this value. Here, we provide evidence that G. sulfurreducens also requires different electron transfer proteins for reduction of a wide range of Fe(III)- and Mn(IV)-(oxyhydr)oxides, and must transition from a high- to low-potential pathway during reduction of commonly studied soluble and insoluble metal electron acceptors...
January 3, 2017: ISME Journal
https://www.readbyqxmd.com/read/28045020/structural-characterisation-of-the-fatty-acid-biosynthesis-enzyme-fabf-from-the-pathogen-listeria-monocytogenes
#13
Tatiana P Soares da Costa, Jeffrey D Nanson, Jade K Forwood
Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.35 Å crystal structure of FabF from L. monocytogenes, providing an excellent platform for the rational design of novel inhibitors...
January 3, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28042595/expression-purification-and-crystallization-of-the-herpesvirus-nuclear-egress-complex-nec
#14
Janna M Bigalke, Ekaterina E Heldwein
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures...
July 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28035668/ice-and-anti-nucleating-activities-of-an-ice-binding-protein-from-the-annual-grass-brachypodium-distachyon
#15
Melissa Bredow, Heather E Tomalty, Lindsay Smith, Virginia K Walker
Plants exposed to sub-zero temperatures face unique challenges that threaten their survival. The growth of ice crystals in the extracellular space can cause cellular dehydration, plasma membrane rupture, and eventual cell death. Additionally, some pathogenic bacteria cause tissue damage by initiating ice crystal growth at high sub-zero temperatures through the use of ice nucleating proteins (INPs), presumably to access nutrients from lysed cells. An annual species of brome grass, Brachypodium distachyon (Bd), produces an ice-binding protein (IBP) that shapes ice with a modest depression of the freezing point (~0...
December 30, 2016: Plant, Cell & Environment
https://www.readbyqxmd.com/read/28034717/crystal-structure-of-a-phospholipase-a2-from-bothrops-asper-venom-insights-into-a-new-putative-myotoxic-cluster
#16
Guilherme H M Salvador, Juliana I Dos Santos, Bruno Lomonte, Marcos R M Fontes
Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s)...
December 26, 2016: Biochimie
https://www.readbyqxmd.com/read/28030602/structural-and-biochemical-characterization-of-chlamydia-trachomatis-dsba-reveals-a-cysteine-rich-and-weakly-oxidising-oxidoreductase
#17
Signe Christensen, Morten K Grøftehauge, Karl Byriel, Wilhelmina M Huston, Emily Furlong, Begoña Heras, Jennifer L Martin, Róisín M McMahon
The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character...
2016: PloS One
https://www.readbyqxmd.com/read/28025687/characterisation-of-the%C3%A2-daacs%C3%A2-family-escherichia-coli-glutamate-aspartate-proton-symporter-gltp-using-computational-chemical-biochemical-and-biophysical-methods
#18
Moazur Rahman, Fouzia Ismat, Li Jiao, Jocelyn M Baldwin, David J Sharples, Stephen A Baldwin, Simon G Patching
Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh...
December 26, 2016: Journal of Membrane Biology
https://www.readbyqxmd.com/read/28018966/crystal-structures-of-mdfa-complexed-with-acetylcholine-and-inhibitor-reserpine
#19
Ming Liu, Jie Heng, Yuan Gao, Xianping Wang
The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family. Two crystal structures were produced for E. coli MdfA, one in complex with acetylcholine and the other with potential reserpine, which are substrate and inhibitor of VNTs, respectively...
2016: Biophysics Reports
https://www.readbyqxmd.com/read/28013221/biochemical-characterization-of-the-flagellar-stator-associated-inner-membrane-protein-flil-from-vibrio-alginolyticus
#20
Ananthanarayanan Kumar, Miyu Isumi, Mayuko Sakuma, Shiwei Zhu, Yuuki Nishino, Yasuhiro Onoue, Seiji Kojima, Yohei Miyanoiri, Katsumi Imada, Michio Homma
The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca...
December 24, 2016: Journal of Biochemistry
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