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  • Journal article
    Benjamin S, Williams F, Kerry L, Matthews Set al., 2015,

    NMR assignment of the immune mapped protein 1 (IMP1) homologue from Plasmodium falciparum

    , Biomolecular NMR Assignments, Vol: 9, Pages: 393-395, ISSN: 1874-2718
  • Journal article
    Hunter PJ, Shaw RK, Berger CN, Frankel G, Pink D, Hand Pet al., 2015,

    Older leaves of lettuce (Lactuca spp.) support higher levels of Salmonella enterica ser. Senftenberg attachment and show greater variation between plant accessions than do younger leaves

    , FEMS Microbiology Letters, Vol: 362, ISSN: 0378-1097

    Salmonella can bind to the leaves of salad crops including lettuce and survive for commercially relevant periods. Previous studies have shown that younger leaves are more susceptible to colonization than older leaves and that colonization levels are dependent on both the bacterial serovar and the lettuce cultivar. In this study, we investigated the ability of two Lactuca sativa cultivars (Saladin and Iceberg) and an accession of wild lettuce (L. serriola) to support attachment of Salmonella enterica serovar Senftenberg, to the first and fifth to sixth true leaves and the associations between cultivar-dependent variation in plant leaf surface characteristics and bacterial attachment. Attachment levels were higher on older leaves than on the younger ones and these differences were associated with leaf vein and stomatal densities, leaf surface hydrophobicity and leaf surface soluble protein concentrations. Vein density and leaf surface hydrophobicity were also associated with cultivar-specific differences in Salmonella attachment, although the latter was only observed in the older leaves and was also associated with level of epicuticular wax.

  • Journal article
    Carrara G, Saraiva N, Parsons M, Byrne B, Prole DL, Taylor CW, Smith GLet al., 2015,

    Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration

    , Journal of Biological Chemistry, Vol: 290, Pages: 11785-11801, ISSN: 0021-9258

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.Background: GAAPs regulate intracellular Ca2+ fluxes, cell migration, and apoptosis.Results: GAAP forms a cation-selective channel, and residues involved in its ion-conducting properties were identified.Conclusion: Mutations within the pore demonstrate that GAAP effects on apoptosis and migration are separable.Significance: Characterization of the pore region of GAAP provides insight into the mechanism of action of this novel and highly conserved ion channel.

  • Journal article
    Sacharz J, Bryan SJ, Yu J, Burroughs NJ, Spence EM, Nixon PJ, Mullineaux CWet al., 2015,

    Sub-cellular location of FtsH proteases in the cyanobacterium Synechocystis sp PCC 6803 suggests localised PSII repair zones in the thylakoid membranes

    , Molecular Microbiology, Vol: 96, Pages: 448-462, ISSN: 1365-2958

    In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub-cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP-tagged versions of its four FtsH proteases. The ftsH2–gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2–GFP patches represent Photosystem II ‘repair zones’ within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti-GFP affinity pull-downs provide the first indication of the composition of the putative repair zones.

  • Journal article
    Cardona Londono T, Murray JW, Rutherford AW, 2015,

    Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria

    , Molecular Biology and Evolution, Vol: 32, Pages: 1310-1328, ISSN: 1537-1719

    Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages towards the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.

  • Journal article
    Shah UV, Amberg C, Diao Y, Yang Z, Heng JYYet al., 2015,

    Heterogeneous nucleants for crystallogenesis and bioseparation

    , CURRENT OPINION IN CHEMICAL ENGINEERING, Vol: 8, Pages: 69-75, ISSN: 2211-3398
  • Journal article
    Serrao E, Ballandras-Colas A, Cherepanov P, Maertens GN, Engelman ANet al., 2015,

    Key determinants of target DNA recognition by retroviral intasomes

    , Retrovirology, Vol: 12, ISSN: 1742-4690

    Background: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joiningand generates a short (4–6 bp) duplication of host DNA flanking the provirus. We previously determined two keyparameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiencyvirus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integrationsites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase.Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings withrespect to length of target site duplication (TSD).Results: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literatureor generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RYdinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated withTSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integrationinto nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization.Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A),which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloneymurine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts withand is catalytically stimulated by cellular bromodomain containing 4 protein.Conclusions: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bondsinto two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bendDNA as sharply as viruses tha

  • Journal article
    Carrara M, Prischi F, Nowak P, Ali Met al., 2015,

    Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling

    , EMBO Journal, Vol: 34, Pages: 1589-1600, ISSN: 0261-4189

    Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein‐folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X‐ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.

  • Journal article
    Aragon IM, Pérez-Mendoza D, Moscoso JA, Faure E, Guery B, Gallegos M-T, Filloux A, Ramos Cet al., 2015,

    Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections

    , Environmental Microbiology, Vol: 17, Pages: 4332-4351, ISSN: 1462-2920

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

  • Journal article
    Taylor JD, Matthews SJ, 2015,

    New insight into the molecular control of bacterial functional amyloids.

    , Frontiers in Cellular and Infection Microbiology, Vol: 5, ISSN: 2235-2988

    Amyloid protein structure has been discovered in a variety of functional or pathogenic contexts. What distinguishes the former from the latter is that functional amyloid systems possess dedicated molecular control systems that determine the timing, location, and structure of the fibers. Failure to guide this process can result in cytotoxicity, as observed in several pathologies like Alzheimer's and Parkinson's Disease. Many gram-negative bacteria produce an extracellular amyloid fiber known as curli via a multi-component secretion system. During this process, aggregation-prone, semi-folded curli subunits have to cross the periplasm and outer-membrane and self-assemble into surface-attached fibers. Two recent breakthroughs have provided molecular details regarding periplasmic chaperoning and subunit secretion. This review offers a combined perspective on these first mechanistic insights into the curli system.

  • Journal article
    Boussac A, Rutherford AW, Sugiura M, 2015,

    Electron transfer pathways from the S<sub>2</sub>-states to the S<sub>3</sub>-states either after a Ca<SUP>2+</SUP>/Sr<SUP>2+</SUP> or a Cl<SUP>-</SUP>/I<SUP>-</SUP> exchange in Photosystem II from

    , BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, Vol: 1847, Pages: 576-586, ISSN: 0005-2728
  • Journal article
    Gross CA, Gruendling A, 2015,

    Editorial overview: Cell regulation: When you think you know it all, there is another layer to be discovered

    , CURRENT OPINION IN MICROBIOLOGY, Vol: 24, Pages: V-VII, ISSN: 1369-5274
  • Journal article
    Liu B, Zhu F, Wu H, Matthews Set al., 2015,

    NMR assignment of the amylase-binding protein A from <i>Streptococcus parasanguinis</i>

    , BIOMOLECULAR NMR ASSIGNMENTS, Vol: 9, Pages: 173-175, ISSN: 1874-2718
  • Journal article
    Cotton CA, Douglass JS, De Causmaecker S, Brinkert K, Cardona T, Fantuzzi A, Rutherford AW, Murray JWet al., 2015,

    Photosynthetic constraints on fuel from microbes.

    , Frontiers in Bioengineering and Biotechnology, Vol: 3, ISSN: 2296-4185
  • Journal article
    Weston DJ, Russell RA, Batty E, Jensen K, Stephens DA, Adams NM, Freemont PSet al., 2015,

    New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts

  • Journal article
    Khurshid S, Govada L, El-Sharif HF, Reddy SM, Chayen NEet al., 2015,

    Automating the application of smart materials for protein crystallization

  • Journal article
    Kaucikas M, Fitzpatrick A, Bryan E, Struve A, Henning R, Kosheleva I, Srajer V, Groenhof G, Van Thor JJet al., 2015,

    Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

  • Journal article
    Corrigan RM, Bowman L, Willis AR, Kaever V, Gruendling Aet al., 2015,

    Cross-talk between Two Nucleotide-signaling Pathways in Staphylococcus aureus

    , Journal of Biological Chemistry, Vol: 290, Pages: 5826-5839, ISSN: 0021-9258

    Nucleotide-signaling pathways are found in all kingdoms oflife and are utilized to coordinate a rapid response to externalstimuli. The stringent response alarmones guanosine tetra(ppGpp)and pentaphosphate (pppGpp) control a globalresponse allowing cells to adapt to starvation conditions such asamino acid depletion. One more recently discovered signalingnucleotide is the secondary messenger cyclic diadenosinemonophosphate (c-di-AMP). Here, we demonstrate that thissignaling nucleotide is essential for the growth of Staphylococcusaureus, and its increased production during late growthphases indicates that c-di-AMP controls processes that areimportant for the survival of cells in stationary phase. By examiningthe transcriptional profile of cells with high levels of c-diAMP,we reveal a significant overlap with a stringent responsetranscription signature. Examination of the intracellular nucleotidelevels under stress conditions provides further evidencethat high levels of c-di-AMP lead to an activation of the stringentresponse through a RelA/SpoT homologue (RSH) enzymedependentincrease in the (p)ppGpp levels. This activation isshown to be indirect as c-di-AMP does not interact directly withthe RSH protein. Our data extend this interconnection furtherby showing that the S. aureus c-di-AMP phosphodiesteraseenzyme GdpP is inhibited in a dose-dependent manner byppGpp, which itself is not a substrate for this enzyme. Altogether,these findings add a new layer of complexity to ourunderstanding of nucleotide signaling in bacteria as they highlightintricate interconnections between different nucleotidesignalingnetworks.

  • Journal article
    Fernandes H, Leen EN, Cromwell H, Pfeil M-P, Curry Set al., 2015,

    Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease-substrate interactions

    , PEERJ, Vol: 3, ISSN: 2167-8359
  • Journal article
    Garnett JA, Muhl D, Douse CH, Hui K, Busch A, Omisore A, Yang Y, Simpson P, Marchant J, Waksman G, Matthews S, Filloux Aet al., 2015,

    Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation

    , Protein Science, Vol: 24, Pages: 670-687, ISSN: 1469-896X

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous withcystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a modelorganism to study biofilms: a bacterial population embedded in an extracellular matrix that provideprotection from environmental pressures and lead to persistence. A number of Chaperone-UsherPathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili onthe bacterial surface. One of these, encoded by the cupB operon, is unique as it contains anonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised ofa C-terminal integral membrane b-barrel pore with tandem N-terminal POTRA (POlypeptide TRansportAssociated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). UsingNMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4(LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 cantranslocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observationthat suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstratethat tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa hasdeveloped synergy between these systems. Furthermore, we have determined the solutionstructureof the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mappingand in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residuesfor TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for eachPOTRA domain.

  • Journal article
    Carrara M, Prischi F, Nowak PR, Kopp MC, Ali MMUet al., 2015,

    Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein C(H)1 to initiate ER stress signaling

    , eLife, Vol: 4, Pages: 1-16, ISSN: 2050-084X

    The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction.

  • Journal article
    Kaucikas M, Tros M, van Thor JJ, 2015,

    Photoisomerization and proton transfer in the forward and reverse photoswitching of the fast-switching M159T mutant of the dronpa fluorescent protein

    , The Journal of Physical Chemistry B: Biophysical Chemistry, Biomaterials, Liquids, and Soft Matter, Vol: 119, Pages: 2350-2362, ISSN: 1520-5207

    The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis–trans and trans–cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright “on” and dark “off” states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the A

  • Journal article
    Shah UV, Parambil JV, Williams DRM, Hinder SJ, Heng Jet al., 2015,

    Preparation and characterisation of 3D nanotemplates for protein crystallisation

    , Powder Technology, Vol: 282, Pages: 10-18, ISSN: 0032-5910

    Heterogeneous template nucleants are gaining pace as a favoured tool for crystallisation of proteins that may be otherwise difficult to crystallise. A systematic understanding on protein-nucleant interactions has to be developed to enable the development of nucleants for a wide spectrum of biological macromolecules. Thorough characterisation of the nucleants is the key starting point to achieve this aim. This report focuses on the method to produce and characterise functionalised 3D nanotemplates with controlled porosity in the range of 3-22. nm and surface chemistry that can vary from highly hydrophilic to highly hydrophobic in nature. BET and TEM are used to study porosity and pore size distribution while contact angle, XPS and zeta potential are used to investigate surface chemistry of the nucleants. These functionalised 3D nanotemplates are hereby reported to produce protein crystals (concanavalin A and catalase) of different habits without changing any other crystallisation parameters other than the surface chemistry of the templates. This emphasises the potential of 3D nanotemplates with well-ordered porosity and chemistry for further development in protein crystallisation experiments.

  • Journal article
    Evans ML, Chorell E, Taylor JD, Aden J, Gotheson A, Li F, Koch M, Sefer L, Matthews SJ, Wittung-Stafshede P, Almqvist F, Chapman MRet al., 2015,

    The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation

    , MOLECULAR CELL, Vol: 57, Pages: 445-455, ISSN: 1097-2765
  • Journal article
    Byrne B, 2015,

    Pichia pastoris as an expression host for membrane protein structural biology

    , Current Opinion in Structural Biology, Vol: 32, Pages: 9-17, ISSN: 0959-440X

    The methylotrophic yeast Pichia pastoris is a widely used recombinant expression host. P. pastoris combines the advantages of ease of use, relatively rapid expression times and low cost with eukaryotic co-translational and post-translational processing systems and lipid composition. The suitability of P. pastoris for high density controlled culture in bioreactors means large amounts of protein can be obtained from small culture volumes. This review details the key features of P. pastoris, which have made it a particularly useful system for the production of membrane proteins, including receptors, channels and transporters, for structural studies. In addition, this review provides an overview of all the constructs and cell strains used to produce membrane proteins, which have yielded high resolution structures.

  • Journal article
    Campeotto I, Zhang Y, Mladenov MG, Freemont PS, Grundling Aet al., 2015,

    Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family

    , Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351X

    Signaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.

  • Journal article
    van Thor JJ, Madsen A, 2015,

    A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

    , Structural Dynamics, Vol: 2, ISSN: 2329-7778

    In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.

  • Journal article
    Gu R-X, Corradi V, Singh G, Beis K, Tieleman Pet al., 2015,

    Conformational Changes of the ABC Transporter McjD Revealed by Molecular Dynamics Simulations

    , BIOPHYSICAL JOURNAL, Vol: 108, Pages: 89A-89A, ISSN: 0006-3495
  • Journal article
    Schreiber F, Kay S, Frankel G, Clare S, Goulding D, van de Vosse E, van Dissel JT, Strugnell R, Thwaites G, Kingsley RA, Dougan G, Baker Set al., 2015,

    The H<i>d</i>, H<i>j</i>, and H<i>z66</i> flagella variants of <i>Salmonella enterica</i> serovar Typhi modify host responses and cellular interactions

    , SCIENTIFIC REPORTS, Vol: 5, ISSN: 2045-2322
  • Journal article
    Huynh M-H, Liu B, Henry M, Liew L, Matthews SJ, Carruthers VBet al., 2015,

    Structural Basis of <i>Toxoplasma gondii</i> MIC2-associated Protein Interaction with MIC2

    , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 290, Pages: 1432-1441

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