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Crystallization of membrane protein

Louise Wogelred, Fredrik Höök, Björn Agnarsson, Peter Sjövall
Accurate detection and quantification of individual molecules is important for the development of improved diagnostic methods as well as biochemical characterization of disease progression and treatments. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique capable of imaging the distribution of specific molecules on surfaces with a high spatial resolution (<1 μm) and high sensitivity. ToF-SIMS is particularly suitable for detection of molecules up to ∼2 kDa, including lipids, whereas larger molecules, such as peptides and proteins, are fragmented during analysis, which makes them difficult to identify...
March 15, 2018: Biointerphases
Benjamin Stauch, Vadim Cherezov
G protein-coupled receptors (GPCRs) represent a large superfamily of membrane proteins that mediate cell signaling and regulate a variety of physiological processes in the human body. Structure-function studies of this superfamily were enabled a decade ago by multiple breakthroughs in technology that included receptor stabilization, crystallization in a membrane environment, and microcrystallography. The recent emergence of X-ray free-electron lasers (XFELs) has further accelerated structural studies of GPCRs and other challenging proteins by overcoming radiation damage and providing access to high-resolution structures and dynamics using micrometer-sized crystals...
March 15, 2018: Annual Review of Biophysics
Dehbia Benkerrou, Matteo Ceccarelli
One of the greatest health threats facing modern medicine is the emergence of new bacterial strains which are increasingly resistant to almost all currently available antibiotics. According to a CDC (Center for Disease Control and Prevention) report published in 2013, 63% of Acinetobacter species have been identified as Multidrug resistant strains. As for other Gram-negative bacteria, the presence of an outer membrane increases the intrinsic resistance of A. baumannii to most antibiotics. The outer membrane of A...
March 15, 2018: Physical Chemistry Chemical Physics: PCCP
Geoffrey Masuyer, Sicai Zhang, Sulyman Barkho, Yi Shen, Linda Henriksson, Sara Košenina, Min Dong, Pål Stenmark
Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6...
March 14, 2018: Scientific Reports
Benedetta Righino, Angelo Minucci, Davide Pirolli, Ettore Capoluongo, Giorgio Conti, Daniele De Luca, Maria Cristina De Rosa
Phospholipase A2-IIA catalyzes the hydrolysis of the sn-2 ester of glycerophospholipids. A rare c.428G > A (p.Arg143His) variant in PLA2G2A gene was found in two infants affected by acute respiratory distress syndrome (ARDS) by whole coding region and exon/intron boundaries sequencing. To obtain insights into the possible molecular effects of the rare R123H mutation in secretory PLA2-IIA (sPLA2-IIA), molecular modelling, molecular dynamics (MD) using principal component analysis (PCA) and continuum electrostatic calculations were conducted on the crystal structure of the wild type protein and on a generated model structure of the R123H mutant...
March 6, 2018: Journal of Molecular Graphics & Modelling
Khoa Nguyen, Alvaro Garcia, Marc-Antoine Sani, Dil Diaz, Vikas Dubey, Daniel Clayton, Giovanni Dal Poggetto, Flemming Cornelius, Richard J Payne, Frances Separovic, Himanshu Khandelia, Ronald J Clarke
The Na+ ,K+ -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available...
March 6, 2018: Biochimica et Biophysica Acta
Jingjing Jiang, Hongling Wen, Miaomiao Chi, Ying Liu, Jingxue Liu, Zhankui Cao, Li Zhao, Yanyan Song, Na Liu, Lianli Chi, Zhiyu Wang
Human parainfluenza virus type 3 (hPIV3) is an important respiratory pathogen that causes the majority of viral pneumonia of infants and young children. hPIV3 can infect host cells through the synergistic action of hemagglutinin-neuraminidase (HN) protein and the homotypic fusion (F) protein on the viral surface. HN protein plays a variety of roles during the virus invasion process, such as promoting viral particles to bind to receptors, cleaving sialic acid, and activating the F protein. Crystal structure research shows that HN tetramer adopted a "heads-down" conformation, at least two heads dimmer on flank of the four-helix bundle stalk, which forms a symmetrical interaction interface...
March 7, 2018: Virus Genes
Paula G Slater, Sebastian E Gutierrez-Maldonado, Katia Gysling, Carlos F Lagos
The corticotropin-releasing factor (CRF) system is a key mediator of the stress response and addictive behavior. The CRF system includes four peptides: The CRF system includes four peptides: CRF, urocortins I-III, CRF binding protein (CRF-BP) that binds CRF with high affinity, and two class B G-protein coupled receptors CRF1 R and CRF2 R. CRF-BP is a secreted protein without significant sequence homology to CRF receptors or to any other known class of protein. Recently, it has been described a potentiation role of CRF-BP over CRF signaling through CRF2 R in addictive-related neuronal plasticity and behavior...
2018: Frontiers in Endocrinology
Raika Yamagiwa, Takuya Kurahashi, Mariko Takeda, Mayuho Adachi, Hiro Nakamura, Hiroyuki Arai, Yoshitsugu Shiro, Hitomi Sawai, Takehiko Tosha
Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N2 O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO2 - ) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study...
February 27, 2018: Biochimica et Biophysica Acta
Peilong Lu, Duyoung Min, Frank DiMaio, Kathy Y Wei, Michael D Vahey, Scott E Boyken, Zibo Chen, Jorge A Fallas, George Ueda, William Sheffler, Vikram Khipple Mulligan, Wenqing Xu, James U Bowie, David Baker
The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable...
March 2, 2018: Science
Emily J Furlong, Hassanul G Choudhury, Fabian Kurth, Anthony P Duff, Andrew E Whitten, Jennifer L Martin
Correct disulfide bond formation is essential for proper folding of many proteins, including bacterial virulence factors. The suppressor of copper sensitivity (Scs) proteins have roles in dithiol/disulfide interchange and the bacterial response to copper stress. Encoded in a four-gene cassette (ScsABCD) present in many Gram-negative bacteria, the Scs proteins are enigmatic and poorly characterised. Here, we show that the periplasmic α domain of the membrane protein ScsB in the Gram-negative bacterium Proteus mirabilis forms a redox relay with the soluble periplasmic protein PmScsC...
February 28, 2018: Journal of Biological Chemistry
Serena Schwenkert, Sophie Dittmer, Jürgen Soll
Import of preproteins into chloroplasts is an essential process, requiring two major multisubunit protein complexes that are embedded in the outer and inner chloroplast envelope membrane. Both the translocon of the outer chloroplast membrane (Toc), as well as the translocon of the inner chloroplast membrane (Tic) have been studied intensively with respect to their individual subunit compositions, functions and regulations. Recent advances in crystallography have increased our understanding of the operation of these proteins in terms of their interactions and regulation by conformational switching...
February 27, 2018: Essays in Biochemistry
Teng Ma, Xiao Liu, Zhifu Cen, Chuan Xin, Mingfeng Guo, Chaoyu Zou, Wenpeng Song, Rou Xie, Kailun Wang, Hong Zhou, Jun Zhang, Zhen Wang, Ce Bian, Kaijun Cui, Jiong Li, Yu-Quan Wei, Jing Li, Xikun Zhou
BACKGROUND: Interleukin-1β (IL-1β) is a pivotal proinflammatory cytokine that is strongly associated with the inflammation of gout. However, the underlying mechanism through which the production of IL-1β is regulated has not been fully elucidated. Our previous work identified that miR-302b had an important immune regulatory role in bacterial lung infections. This study was conducted to evaluate the function of miR-302b on monosodium urate (MSU) crystal-induced inflammation and its mechanism...
February 26, 2018: Arthritis Research & Therapy
Michela Ciabocco, Patrizia Cancemi, Maria Luisa Saladino, Eugenio Caponetti, Rosa Alduina, Mario Berrettoni
This paper deals with the synthesis and characterization of iron-hexacyanocobaltate (FeHCC) and its antibacterial properties. The nanoparticles were prepared by a facile co-precipitation technique. Crystal structure, particle morphology, and elemental composition were determined using X-ray Powder Diffraction, X-ray fluorescence spectroscopy, Transmission Electron Microscopy (TEM), and Infrared Spectroscopy (IR). The antibacterial activity of the FeHCC nanoparticles was tested against Escherichia coli and Staphylococcus aureus as models for Gram-negative and Gram-positive bacteria, respectively, by bacterial counting method and microscopic visualization (TEM, FEG-SEM, and fluorescence microscopy)...
February 24, 2018: Journal of Biological Inorganic Chemistry: JBIC
Yingwang Ye, Na Ling, Jina Gao, Xiyan Zhang, Maofeng Zhang, Liaowang Tong, Haiyan Zeng, Jumei Zhang, Qingping Wu
Cronobacter sakazakii is an important foodborne pathogen associated with rare but severe infections through consumption of powdered infant formula. Tolerance to osmotic stress in Cronobacter has been described. However, the detailed factors involved in tolerance to osmotic stress in C. sakazakii are poorly understood. In this study, roles of outer membrane protein W (OmpW) on survival rates, morphologic changes of cells, and biofilm formation in C. sakazakii under different NaCl concentrations between wild type (WT) and OmpW mutant (ΔOmpW) were determined...
February 21, 2018: Journal of Dairy Science
Mariam El-Khatib, Chady Nasrallah, Julie Lopes, Que-Tien Tran, Guillaume Tetreau, Hind Basbous, Daphna Fenel, Benoit Gallet, Mathilde Lethier, Jean-Michel Bolla, Jean-Marie Pagès, Michel Vivaudou, Martin Weik, Mathias Winterhalter, Jacques-Philippe Colletier
The gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact, before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, we hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii , Omp-Pst1 and Omp-Pst2. Our crystal structures reveal that these porins can self-associate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities...
February 23, 2018: Proceedings of the National Academy of Sciences of the United States of America
Konstantin R Malley, Olga Koroleva, Ian Miller, Ruslan Sanishvili, Christopher M Jenkins, Richard W Gross, Sergey Korolev
Calcium-independent phospholipase A2 β (iPLA2 β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2 β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2 β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer...
February 22, 2018: Nature Communications
Jeremy M Schieferstein, Ashtamurthy S Pawate, Michael J Varel, Sudipto Guha, Ieva Astrauskaite, Robert B Gennis, Paul J A Kenis
Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure-function relationships. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X-ray transparent microfluidic chips...
February 22, 2018: Lab on a Chip
Takao Hashiguchi, Yoshinari Fukuda, Rei Matsuoka, Daisuke Kuroda, Marie Kubota, Yuta Shirogane, Shumpei Watanabe, Kouhei Tsumoto, Daisuke Kohda, Richard Karl Plemper, Yusuke Yanagi
Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity...
February 20, 2018: Proceedings of the National Academy of Sciences of the United States of America
Jacqueline Kalms, Andrea Schmidt, Stefan Frielingsdorf, Tillmann Utesch, Guillaume Gotthard, David von Stetten, Peter van der Linden, Antoine Royant, Maria Andrea Mroginski, Philippe Carpentier, Oliver Lenz, Patrick Scheerer
[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2 , a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha , is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack...
February 20, 2018: Proceedings of the National Academy of Sciences of the United States of America
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