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  • 1. Beatriz Badia, Mariana
    et al.
    Mans, Robert
    Lis, Alicia V.
    Ariel Tronconi, Marcos
    Lucia Arias, Cintia
    Maurino, Veronica Graciela
    Santiago Andreo, Carlos
    Fabiana Drincovich, Maria
    van Maris, Antonius J. A.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gerrard Wheeler, Mariel Claudia
    Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 4, p. 654-665Article in journal (Refereed)
    Abstract [en]

    NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2, and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc(-)) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc(-) S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2. On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc(-) strains from C-4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and over-expressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C-4 compounds and redox shuttling in plant cells.

  • 2. Brown, Christian
    et al.
    Szpryngiel, Scarlett
    Guanglin, Kuang
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Srivastava, Vaibhav
    Ye, Weihua
    McKee, Lauren S.
    Tu, Yaoquan
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Maler, Lena
    Bulone, Vincent
    Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 16, p. 3072-3088Article in journal (Refereed)
    Abstract [en]

    Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. DatabaseStructural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number .

  • 3. Bulut, Z.
    et al.
    Tepeli, C.
    Nizamlioglu, M.
    Kurar, E.
    Erdogan, M.
    Yilmaz, A.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microsatellite analysis of Kangal shepherd dogs in Turkey, Uzbekistan, Ajerbaijan and Iran2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, p. 349-349Article in journal (Other academic)
  • 4. Fontana, Jacopo M.
    et al.
    Burlaka, Ievgeniia
    Khodus, Georgiy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Calcium oscillations triggered by cardiotonic steroids2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, p. 5450-5455Article, review/survey (Refereed)
    Abstract [en]

    Na+, K+-ATPase (NKA) is well known for its function as an ion pump. Studies during the last decade have revealed an additional role for NKA as a signal transducer. In this brief review, we describe how cardiotonic steroids, which are highly specific NKA ligands, trigger slow Ca2+ oscillations by promoting the interaction between NKA and the inositol trisphosphate receptor, and how this Ca2+ signal activates the NF-B subunit p65 and increases the expression of the antiapoptotic factor Bcl-xL. The potential tissue-protective effects of this signal are discussed.

  • 5.
    Hendrikse, N. M.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry.
    Stromberg, P.
    Nordling, E.
    Syrén, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Redesign of biosynthetic enzymes using ancestral sequence reconstruction2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, p. 86-87Article in journal (Refereed)
  • 6. Henricson, Anna
    et al.
    Käll, Lukas
    Sonnhammer, Erik L. L.
    A novel transmembrane topology of presenilin based on reconciling experimental and computational evidence2005In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, no 11, p. 2727-2733Article in journal (Refereed)
    Abstract [en]

    The transmembrane topology of presenilins is still the subject of debate despite many experimental topology studies using antibodies or gene fusions. The results from these studies are partly contradictory and consequently several topology models have been proposed. Studies of presenilin-interacting proteins have produced further contradiction, primarily regarding the location of the C-terminus. It is thus impossible to produce a topology model that agrees with all published data on presenilin. We have analyzed the presenilin topology through computational sequence analysis of the presenilin family and the homologous presenilin-like protein family. Members of these families are intramembrane-cleaving aspartyl proteases. Although the overall sequence homology between the two families is low, they share the conserved putative active site residues and the conserved 'PAL' motif. Therefore, the topology model for the presenilin-like proteins can give some clues about the presenilin topology. Here we propose a novel nine-transmembrane topology with the C-terminus in the extracytosolic space. This model has strong support from published data on gamma-secretase function and presenilin topology. Contrary to most presenilin topology models, we show that hydrophobic region X is probably a transmembrane segment. Consequently, the C-terminus would be located in the extracytosolic space. However, the last C-terminal amino acids are relatively hydrophobic and in conjunction with existing experimental data we cannot exclude the possibility that the extreme C-terminus could be buried within the gamma-secretase complex. This might explain the difficulties in obtaining consistent experimental evidence regarding the location of the C-terminal region of presenilin.

  • 7. Hrmova, Maria
    et al.
    Farkas, Vladimir
    Harvey, Andrew J.
    Lahnstein, Jelle
    Wischmann, Bente
    Kaewthai, Nomchit
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ezcurra, Inés
    KTH, School of Biotechnology (BIO), Glycoscience.
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fincher, Geoffrey B.
    Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, p. 437-456Article in journal (Refereed)
    Abstract [en]

    A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.

  • 8. Kujawa, Magdalena
    et al.
    Volc, Jindrich
    Halada, Petr
    Sedmera, Petr
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sygmund, Christoph
    Leitner, Christian
    Peterbauer, Clemens
    Haltrich, Dietmar
    Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, p. 879-894Article in journal (Refereed)
    Abstract [en]

    We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K-m). The enzyme may play a role in lignocellulose degradation.

  • 9. Nizamlioglu, M.
    et al.
    Bulut, Z.
    Erdogan, M.
    Tepeli, C.
    Yilmaz, A.
    Kurar, E.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genetic characterization of Akbas shepherd dogs in Turkey, Uzbekistan and Iran using STR markers2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, p. 349-349Article in journal (Other academic)
  • 10.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 11, p. 2668-2676Article, review/survey (Refereed)
    Abstract [en]

    In recent years, classical antibody-based affinity reagents have been challenged by novel types of binding proteins developed by combinatorial protein engineering principles. One of these classes of binding proteins of non-Ig origin are the so-called affibody binding proteins, functionally selected from libraries of a small (6 kDa), non-cysteine three-helix bundle domain used as a scaffold. During the first 10 years since they were first described, high-affinity affibody binding proteins have been selected towards a large number of targets for use in a variety of applications, such as bioseparation, diagnostics, functional inhibition, viral targeting and in vivo tumor imaging/therapy. The small size offers the possibility to produce functional affibody binding proteins also by chemical synthesis production routes, which has been found to be advantageous for the site-specific introduction of various labels and radionuclide chelators.

  • 11. Parmryd, I.
    et al.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Consequences of membrane topography2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 12, p. 2775-2784Article, review/survey (Refereed)
    Abstract [en]

    The surface of mammalian cells is neither smooth nor flat and cells have several times more plasma membrane than the minimum area required to accommodate their shape. We discuss the biological function of this apparent excess membrane that allows the cells to migrate and undergo shape changes and probably plays a role in signal transduction. Methods for studying membrane folding and topography - atomic force microscopy, scanning ion conductance microscopy, fluorescence polarization microscopy and linear dichroism - are described and evaluated. Membrane folding and topography is frequently ignored when interpreting microscopy data. This has resulted in several misconceptions regarding for instance colocalization, membrane organization and molecular clustering. We suggest simple ways to avoid these pitfalls and invoke Occam's razor - that simple explanations are preferable to complex ones. Topography, i.e. deviations from a smooth surface, should always be ruled out as the cause of anomalous data before other explanations are presented. Cell membranes are convoluted into submicrometer ruffles, which are impossible to resolve by most experimental techniques. Here we discuss the importance of considering such subresolution membrane organization when interpreting experimental data and give some examples of techniques where membrane topography can be probed. Finally we speculate what consequences ruffling could have for cellular processes, e. g. receptor signaling.

  • 12. Perriman, Adam W.
    et al.
    Apponyi, Margit A.
    Buntine, Mark A.
    Jackway, Rebecca J.
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface Chemistry.
    White, John W.
    Bowie, John H.
    Surface movement in water of splendipherin, the aquatic male sex pheromone of the tree frog Litoria splendida2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 13, p. 3362-3374Article in journal (Refereed)
    Abstract [en]

    The aquatic sex pheromone splendipherin (GLVSSIGKALGGLLADVVKSKGQPA-OH) of the male green tree frog Litoria splendida moves across the surface of water to reach the female. Surface pressure and X-ray reflectometry measurements confirm that splendipherin is a surface-active molecule, and are consistent with it having an ordered structure, whereby the hydrophilic portion of the peptide interacts with the underlying water and the hydrophobic region is adjacent to the vapour phase. The movement of splendipherin over the surface of water is caused by a surface pressure gradient. In order to better define the structure of splendipherin at the water/air interface we used 2D NMR studies of the pheromone with the solvent system trifluoroethanol/water (1 : 1 v/v). In this solvent system, splendipherin adopts a bent alpha helix from residues V3 to K21. The bending of the helix occurs in the centre of the peptide in the vicinity of G11 and G12. The region of splendipherin from V3 to G11 has well-defined amphipathicity, whereas the amphipathicity from G12 to A25 is reduced by K19 and P24 intruding into the hydrophobic and hydrophilic regions respectively. A helical structure is consistent with X-ray reflectometry data.

  • 13. Podkorytov, I.
    et al.
    Belousov, M.
    Bondarev, S.
    KTH, School of Chemical Science and Engineering (CHE).
    Kampf, K.
    Zhouravleva, G.
    Dvinskikh, Sergey
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Skrynnikov, N.
    Detection of flexible portion of protein chain in Sup35NM amyloid fibrils by means of diffusion-filtered NMR experiment2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, p. 215-215Article in journal (Refereed)
  • 14.
    Sarr, Medoune
    et al.
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Kronqvist, Nina
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Chen, Gefei
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Aleksis, Rihards
    Latvian Inst Organ Synth, Dept Phys Organ Chem, Riga, Latvia..
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Jaudzems, Kristaps
    Latvian Inst Organ Synth, Dept Phys Organ Chem, Riga, Latvia..
    Rising, Anna
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden.;Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Johansson, Jan
    Karolinska Inst, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 10, p. 1873-1885Article in journal (Refereed)
    Abstract [en]

    Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide 17. The fusion protein NT*-17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that 17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, 17 adopts a -sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.

  • 15. Schmidt, H.
    et al.
    Cho, K. H.
    Jacobsen, Elling W.
    KTH, School of Electrical Engineering (EES), Automatic Control.
    Identification of small scale biochemical networks based on general type system perturbations2005In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, no 9, p. 2141-2151Article in journal (Refereed)
    Abstract [en]

    New technologies enable acquisition of large data-sets containing genomic, proteomic and metabolic information that describe the state of a cell. These data-sets call for systematic methods enabling relevant information about the inner workings of the cell to be extracted. One important issue at hand is the understanding of the functional interactions between genes, proteins and metabolites. We here present a method for identifying the dynamic interactions between biochemical components within the cell, in the vicinity of a steady-state. Key features of the proposed method are that it can deal with data obtained under perturbations of any system parameter, not only concentrations of specific components, and that the direct effect of the perturbations does not need to be known. This is important as concentration perturbations are often difficult to perform in biochemical systems and the specific effects of general type perturbations are usually highly uncertain, or unknown. The basis of the method is a linear least-squares estimation, using time-series measurements of concentrations and expression profiles, in which system states and parameter perturbations are estimated simultaneously. An important side-effect of also employing estimation of the parameter perturbations is that knowledge of the system's steady-state concentrations, or activities, is not required and that deviations from steady-state prior to the perturbation can be dealt with. Time derivatives are computed using a zero-order hold discretization, shown to yield significant improvements over the widely used Euler approximation. We also show how network interactions with dynamics that are too fast to be captured within the available sampling time can be determined and excluded from the network identification. Known and unknown moiety conservation relationships can be processed in the same manner. The method requires that the number of samples equals at least the number of network components and, hence, is at present restricted to relatively small-scale networks. We demonstrate herein the performance of the method on two small-scale in silico genetic networks.

  • 16. Spadiut, Oliver
    et al.
    Leitner, Christian
    Salaheddin, Clara
    Varga, Balazs
    Vertessy, Beata G.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Glycoscience.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Haltrich, Dietmar
    Improving thermostability and catalytic activity of pyranose 2-oxidase from Trametes multicolor by rational and semi-rational design2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 3, p. 776-792Article in journal (Refereed)
    Abstract [en]

    The fungal homotetrameric flavoprotein pyranose 2-oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 degrees C, respectively, relative to the wild-type enzyme. Although both wild-type and single mutants showed first-order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.

  • 17.
    Spadiut, Oliver
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Tan, Tien-Chye
    KTH, School of Biotechnology (BIO), Biochemistry.
    Pisanelli, Ines
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry.
    Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 13, p. 2892-2909Article in journal (Refereed)
    Abstract [en]

    Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment (454FSY456) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp452, Phe454 and Tyr456) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

  • 18.
    Spielmann, Thiemo
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Gad, Annica
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Johansson, Sofia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Transient state microscopy probes patterns of altered oxygen consumption in cancer cells2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 5, p. 1317-1332Article in journal (Refereed)
    Abstract [en]

    Altered cellular metabolism plays an important role in many diseases, not least in many forms of cancer, where cellular metabolic pathways requiring lower oxygen consumption are often favored (the so-called Warburg effect). In this work, we have applied fluorescence-based transient state imaging and have exploited the environment sensitivity of long-lived dark states of fluorophores, in particular triplet state decay rates, to image the oxygen consumption of living cells. Our measurements can resolve differences in oxygen concentrations between different regions of individual cells, between different cell types, and also based on what metabolic pathways the cells use. In MCF-7 breast cancer cells, higher oxygen consumption can be detected when they rely on glutamine instead of glucose as their main metabolite, predominantly undergoing oxidative phosphorylation rather than glycolysis. By use of the high triplet yield dye Eosin Y the irradiance requirements during the measurements can be kept low. This reduces the instrumentation requirements, and harmful biological effects from high excitation doses can be avoided. Taken together, our imaging approach is widely applicable and capable of detecting subtle changes in oxygen consumption in live cells, stemming from the Warburg effect or reflecting other differences in the cellular metabolism. This may lead to new diagnostic means as well as advance our understanding of the interplay between cellular metabolism and major disease categories, such as cancer.

  • 19.
    Svedendahl Humble, Maria
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Engelmark Cassimjee, Karim
    KTH, School of Biotechnology (BIO), Biochemistry.
    Håkansson, Maria
    Kimbung, Yengo R
    Walse, Björn
    Abedi, Vahak
    Federsel, Hans-Jürgen
    Berglund, Per
    KTH, School of Biotechnology (BIO), Biochemistry.
    Logan, Derek T
    Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, p. 779-792Article in journal (Refereed)
    Abstract [en]

    The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: •  -transaminases and -transaminases bind by dynamic light scattering (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction) •  -transaminase and -transaminase bind by x-ray crystallography (View interaction).

  • 20. Syren, Per-Olof
    The solution of nitrogen inversion in amidases2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 13, p. 3069-3083Article in journal (Refereed)
    Abstract [en]

    An important mechanistic aspect of enzyme-catalyzed amide bond hydrolysis is the specific orientation of the lone pair of the N atom of the scissile amide bond during catalysis. As discussed in the literature during the last decades, stereoelectronic effects cause the single lone pair in the formed tetrahedral intermediate to be situated in a non-productive conformation in the enzyme active site and hence N atom inversion or rotation is necessary. By discussing recent mechanistic findings in the literature relevant for the conformation of the lone pair of the reacting amide N atom, it is demonstrated that Nature has evolved at least 2 catalytic strategies to cope with the stereoelectronic constraints inherent to amide bond hydrolysis regardless of the fold or catalytic mechanism. One soln. to the inversion problem is to stabilize the transition state of inversion by H-bond formation; another is to introduce a concerted proton shuttle mechanism that avoids inversion and delivers a hydrogen to the lone pair. Here, by using mol. modeling it was demonstrated that the H-bond strategy is general and can be expanded to include many amidases/proteases with important metabolic functions, including the proteasome. Some examples of the proton shuttle mechanism are also mentioned. To complete the picture of efficient enzyme-catalyzed amide bond hydrolysis, general interactions in the active site of these catalysts were discussed. An expanded knowledge of the prerequisites of efficient amide bond hydrolysis beyond the oxyanion hole and the catalytic dyad/triad will be of importance for enzyme and drug design. [on SciFinder(R)]

  • 21.
    Tegel, Hanna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Ottosson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Enhancing the protein production levels in Escherichia coli with a strong promoter2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 5, p. 729-739Article in journal (Refereed)
    Abstract [en]

    In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.

  • 22.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Braun, Laura J.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Aspenstrom, Pontus
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Gad, Annica K. B.
    Univ Madeira, CQM, Campus Penteada, P-9020105 Funchal, Portugal..
    Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 9, p. 1635-1652Article in journal (Refereed)
    Abstract [en]

    Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

  • 23.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Aspenstrom, Pontus
    Braun, Laura J.
    Gad, Annica K. B.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Resolution, target density and labeling effects in colocalization studies - suppression of false positives by nanoscopy and modified algorithms2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 5, p. 882-898Article in journal (Refereed)
    Abstract [en]

    Colocalization analyses of fluorescence images are extensively used to quantify molecular interactions in cells. In recent years, fluorescence nanoscopy has approached resolutions close to molecular dimensions. However, the extent to which image resolution influences different colocalization estimates has not been systematically investigated. In this work, we applied simulations and resolution-tunable stimulated emission depletion microscopy to evaluate how the resolution, molecular density and label size of targeted molecules influence estimates of the most commonly used colocalization algorithms (Pearson correlation coefficient, Manders' M1 and M2 coefficients), as well as estimates by the image cross-correlation spectroscopy method. We investigated the practically measureable extents of colocalization for stimulated emission depletion microscopy with positive and negative control samples with an aim to identifying the strengths and weaknesses of nanoscopic techniques for colocalization studies. At a typical optical resolution of a confocal microscope (200-300 nm), our results indicate that the extent of colocalization is typically overestimated by the tested algorithms, especially at high molecular densities. Only minor effects of this kind were observed at higher resolutions (< 60 nm). By contrast, underestimation of colocalization may occur if the resolution is close to the size of the label/affinity molecules themselves. To suppress false positives at confocal resolutions and high molecular densities, we introduce a statistical variant of Costes' threshold searching algorithm, used in combination with correlation-based methods like the Pearson coefficient and the image cross-correlation spectroscopy approach, to set intensity thresholds separating background noise from signals.

  • 24. Zelenin, S.
    et al.
    Illarionova, N.
    Kamali-Zare, P.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, A.
    Identification and functional significance of a brain AQP4/Na+, K+-ATPase/mGluR5 macromolecular complex2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, p. 207-207Article in journal (Refereed)
1 - 24 of 24
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