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  • Journal article
    Curry S, 2014,

    Open access - reasons to be cheerful: a reply to Agrawal

    , Trends in Plant Science, Vol: 19, Pages: 196-197

    Anurag Agrawal's recent letter on open access publishing raises an important topic that many researchers may have found difficult to engage with, not least because its myriad complexities are frequently enveloped in strong cross-currents of opinion. Agrawal is concerned that some scientists might still be rather uncritical of the accelerating open-access bandwagon and rightly highlights some of the possible pitfalls. However, although it is important to be aware of the risks of open access, Agrawal was more pessimistic in his assessment than is warranted by the evidence and, in my view, paid insufficient attention to the possible benefits.

  • Journal article
    Prischi F, Nowak PR, Carrara M, Ali MMUet al., 2014,

    Phosphoregulation of Ire1 RNase splicing activity.

    , Nat Commun, Vol: 5

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation.

  • Journal article
    Serrao E, Krishnan L, Shun M-C, Li X, Cherepanov P, Engelman A, Maertens GNet al., 2014,

    Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding

    , Nucleic Acids Research, Vol: 42, Pages: 5164-5176, ISSN: 0305-1048

    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration.

  • Journal article
    Liu B, Shadrin A, Sheppard C, Mekler V, Xu Y, Severinov K, Matthews S, Wigneshweraraj Set al., 2014,

    A bacteriophage transcription regulator inhibits bacterial transcription initiation by Sigma-factor displacement

    , Nucleic Acids Research, Vol: 42, Pages: 4294-4305, ISSN: 0305-1048

    Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription.

  • Journal article
    Bubeck D, 2014,

    The Making of a Macromolecular Machine: Assembly of the Membrane Attack Complex

    , BIOCHEMISTRY, Vol: 53, Pages: 1908-1915, ISSN: 0006-2960
  • Journal article
    Sharma A, Leach RN, Gell C, Zhang N, Burrows PC, Shepherd DA, Wigneshweraraj S, Smith DA, Zhang X, Buck M, Stockley PG, Tuma Ret al., 2014,

    Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies

    , NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 5177-5190, ISSN: 0305-1048
  • Journal article
    Montalvao R, Camilloni C, De Simone A, Vendruscolo Met al., 2014,

    New opportunities for tensor-free calculations of residual dipolar couplings for the study of protein dynamics

    , JOURNAL OF BIOMOLECULAR NMR, Vol: 58, Pages: 233-238, ISSN: 0925-2738
  • Journal article
    Reichmann NT, Cassona CP, Monteiro JM, Bottomley AL, Corrigan RM, Foster SJ, Pinho MG, Gruendling Aet al., 2014,

    Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in <i>Staphylococcus aureus</i>

    , MOLECULAR MICROBIOLOGY, Vol: 92, Pages: 273-286, ISSN: 0950-382X
  • Journal article
    Ball G, Filloux A, Voulhoux R, 2014,

    A method to capture large DNA fragments from genomic DNA.

    , Methods Mol Biol, Vol: 1149, Pages: 491-500

    The gene capture technique is a powerful tool that allows the cloning of large DNA regions (up to 80 kb), such as entire genomic islands, without using restriction enzymes or DNA amplification. This technique takes advantage of the high recombinant capacity of the yeast. A "capture" vector containing both ends of the target DNA region must first be constructed. The target region is then captured by co-transformation and recombination in yeast between the "capture" vector and appropriate genomic DNA. The selected recombinant plasmid can be verified by sequencing and transferred in the bacteria for multiple applications. This chapter describes a protocol specifically adapted for Pseudomonas aeruginosa genomic DNA capture.

  • Conference paper
    Murray JW, 2014,

    Protein design for artificial photosynthesis

    , 247th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
  • Conference paper
    Thompson LM, Champion PM, Sage JT, Frisch MJ, van Thor JJ, Bearpark MJet al., 2014,

    Analytical harmonic vibrational frequencies for the green fluorescent protein computed with ONIOM: Chromophore mode character and its response to environment

    , 247th National Spring Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
  • Journal article
    Joshi A, Esteve V, Buckroyd AN, Blatter M, Allain FH-T, Curry Set al., 2014,

    Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB)

    , PEERJ, Vol: 2, ISSN: 2167-8359
  • Journal article
    Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A, Fronzes R, Orlova EV, Waksman Get al., 2014,

    Structure of a Type IV secretion system

    , Nature, Vol: 508, Pages: 550-553, ISSN: 0028-0836

    Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells1,2, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells3. Given the complex choreography of the substrate through the secretion apparatus4, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems1,4,5,6.

  • Journal article
    Bertheleme N, Strege A, Bunting SE, Dowell SJ, Byrne Bet al., 2014,

    Arginine 199 and leucine 208 have key roles in the control of adenosine A(2A) receptor signalling function

    , PLoS One, Vol: 9, Pages: 1-10, ISSN: 1932-6203

    One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant) and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.

  • Journal article
    Knoppova J, Sobotka R, Tichy M, Yu J, Konik P, Halada P, Nixon PJ, Komenda Jet al., 2014,

    Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in synechocystis

    , The Plant Cell, Vol: 26, Pages: 1200-1212, ISSN: 1040-4651

    Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.

  • Journal article
    Yeung HO, Foerster A, Bebeacua C, Niwa H, Ewens C, McKeown C, Zhang X, Freemont PSet al., 2014,

    Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

    , Open Biology, Vol: 4, ISSN: 2046-2441

    The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.

  • Journal article
    Craig R, Lee KH, Mun JY, Torre I, Luther PKet al., 2014,

    Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C

    , PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, Vol: 466, Pages: 425-431, ISSN: 0031-6768
  • Journal article
    Wlodarska M, Thaiss CA, Nowarski R, Henao-Mejia J, Zhang J-P, Brown EM, Frankel G, Levy M, Katz MN, Philbrick WM, Elinav E, Finlay BB, Flavell RAet al., 2014,

    NLRP6 Inflammasome Orchestrates the Colonic Host-Microbial Interface by Regulating Goblet Cell Mucus Secretion

    , CELL, Vol: 156, Pages: 1045-1059, ISSN: 0092-8674
  • Journal article
    Gumbart JC, Beeby M, Jensen GJ, Roux Bet al., 2014,

    Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale aimulations

    , PLOS Computational Biology, Vol: 10, ISSN: 1553-734X

    Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgorpressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterialcell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid,yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by themolecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is notknown. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in adisordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as knownexperimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other featuressuch as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting thedisordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure andbehavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of allatomsimulations to reproduce a range of macroscopic observables for extended polymer meshes.

  • Journal article
    Maertens GN, Cook NJ, Wang W, Hare S, Gupta SS, Oeztop I, Lee K, Pye VE, Cosnefroy O, Snijders AP, KewalRamani VN, Fassati A, Engelman A, Cherepanov Pet al., 2014,

    Structural basis for nuclear import of splicing factors by human transportin 3

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 111, Pages: 2728-2733, ISSN: 0027-8424

    Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3–CPSF6 nexus in HIV-1 biology.

  • Journal article
    Riese SB, Kuehne C, Tedder TF, Hallmann R, Hohenester E, Buscher Ket al., 2014,

    Heterotropic Modulation of Selectin Affinity by Allosteric Antibodies Affects Leukocyte Rolling

    , JOURNAL OF IMMUNOLOGY, Vol: 192, Pages: 1862-1869, ISSN: 0022-1767
  • Journal article
    Lansky S, Alalouf O, Solomon HV, Alhassid A, Govada L, Chayen NE, Belrhali H, Shoham Y, Shoham Get al., 2014,

    A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from <i>Geobacillus stearothermophilus</i>

  • Conference paper
    Hirt MN, Boeddinghaus J, Schaaf S, Mitchell A, Eder A, Luther PK, Boernchen C, Stenzig J, Hansen A, Eschenhagen Tet al., 2014,

    Maturation of Engineered Heart Tissue (EHT) by Permanent Electrical Stimulation

    , 80th Annual Meeting of the Deutsche-Gesellschaft-fur-Experimentelle-und-Klinische-Pharmakologie-und-Toxikologie-e-V, Publisher: SPRINGER, Pages: S50-S50, ISSN: 0028-1298
  • Journal article
    Thompson LM, Lasoroski A, Champion PM, Sage JT, Frisch MJ, van Thor JJ, Bearpark MJet al., 2014,

    Analytical Harmonic Vibrational Frequencies for the Green Fluorescent Protein Computed with ONIOM: Chromophore Mode Character and Its Response to Environment

    , Journal of Chemical Theory and Computation, Vol: 10, Pages: 751-766, ISSN: 1549-9618
  • Conference paper
    Toepfer C, Sikkel M, Caorsi V, Copeland O, Martinez IT, West T, Marston S, Luther P, Lyon A, Macleod K, Ferenczi Met al., 2014,

    Effects of Chronic Myocardial Infarction on Cardiac Muscle Performance and Structure In-Vivo and In-Vitro

    , 58th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 343A-344A, ISSN: 0006-3495
  • Journal article
    Lee K-Y, Blaker JJ, Murakami R, Heng JYY, Bismarck Aet al., 2014,

    Phase Behavior of Medium and High Internal Phase Water-in-Oil Emulsions Stabilized Solely by Hydrophobized Bacterial Cellulose Nanofibrils

    , LANGMUIR, Vol: 30, Pages: 452-460, ISSN: 0743-7463
  • Journal article
    Roqué Rosell NR, Mokhlesi L, Milton NE, Sweeney TR, Zunszain PA, Curry S, Leatherbarrow RJet al., 2014,

    Design and synthesis of irreversible inhibitors of foot-and-mouth disease virus 3C protease.

    , Bioorg Med Chem Lett, Vol: 24, Pages: 490-494

    Foot-and-mouth disease virus (FMDV) causes a highly infectious and economically devastating disease of livestock. The FMDV genome is translated as a single polypeptide precursor that is cleaved into functional proteins predominantly by the highly conserved viral 3C protease, making this enzyme an attractive target for antiviral drugs. A peptide corresponding to an optimal substrate has been modified at the C-terminus, by the addition of a warhead, to produce irreversible inhibitors that react as Michael acceptors with the enzyme active site. Further investigation highlighted key structural determinants for inhibition, with a positively charged P2 being particularly important for potency.

  • Journal article
    Fusco D, Headd JJ, De Simone A, Wang J, Charbonneau Pet al., 2014,

    Characterizing protein crystal contacts and their role in crystallization: rubredoxin as a case study

    , SOFT MATTER, Vol: 10, Pages: 290-302, ISSN: 1744-683X
  • Journal article
    Helaine S, Cheverton AM, Watson KG, Faure LM, Matthews SA, Holden DWet al., 2014,

    Internalization of salmonella by macrophages induces formation of nonreplicating persisters

    , SCIENCE, Vol: 343, Pages: 204-208, ISSN: 0036-8075

    Many bacterial pathogens cause persistent infections despite repeated antibiotic exposure. Bacterial persisters are antibiotic-tolerant cells, but little is known about their growth status and the signals and pathways leading to their formation in infected tissues. We used fluorescent single-cell analysis to identify Salmonella persisters during infection. These were part of a nonreplicating population formed immediately after uptake by macrophages and were induced by vacuolar acidification and nutritional deprivation, conditions that also induce Salmonella virulence gene expression. The majority of 14 toxin-antitoxin modules contributed to intracellular persister formation. Some persisters resumed intracellular growth after phagocytosis by naïve macrophages. Thus, the vacuolar environment induces phenotypic heterogeneity, leading to either bacterial replication or the formation of nonreplicating persisters that could provide a reservoir for relapsing infection.

  • Conference paper
    Hedberg S, Quigley A, Heng JYY, Williams DR, Liddell Jet al., 2014,

    Self-interaction chromatography (SIC) of mabs: New methods for estimating the dead volume in SIC and using sic to predict mab stability

    , Pages: 897-907

    Protein-protein molecular interactions are known to be involved in protein solution aggregation behaviour; however the mechanisms leading to protein aggregation are still not fully understood. The osmotic second virial coefficient (B22) is a fundamental physiochemical property that describes proteinprotein interactions in solution, which can be a useful tool to predict aggregation propensity of proteins. This work includes two experimental SIC studies on both model proteins and therapeutic mAbs of different sizes. The first study is an evaluation of two different experimental techniques used to determine SIC dead volumes and the second study uses SIC results for mAb to predict stability. Accurate dead retention volumes are essential for the accurate determinations of B22. The traditional method of estimating dead volume for SIC includes the use of a dead column (without protein immobilised) where the retention volume for proteins can be established. For this technique the dead volume was established for the proteins over a wide range of solution conditions (pH and salt concentrations), and then compared with a new method, where a number of non-interacting dextrans of different molecular weights (MW) (including the MW's of the protein) were employed to find the dead retention volume. The results for the traditional technique with a dead column changed depending on the protein used; only certain model proteins kept a constant dead retention volume when the pH was changing under a constant high salt concentration to minimise protein-surface interactions. Several proteins, including the mAb, exhibited an increased dead retention volume especially when exposed to lower pH. From this it can be concluded that there is no absolute dead volume that can be determined by this technique which are independent of solution conditions. The new technique involving dextrans gives a better overall result for the dead volume for proteins such as mAbs. The second study shows that the SI

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