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  • 1.
    Aguilar, Ximena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e98575-Article in journal (Refereed)
    Abstract [en]

    Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.

  • 2.
    Augusti, Angela
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Betson, Tatiana R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Deriving correlated climate and physiological signals from deuterium isotopomers in tree rings2008In: Chemical Geology, ISSN 0009-2541, E-ISSN 1872-6836, Vol. 252, no 1-2, p. 1-8Article in journal (Refereed)
    Abstract [en]

    he deuterium (D) abundance of tree-ring cellulose archives past climatic conditions, but previous attempts to access this archive have led to conflicting results. Based on an overview of D fractionation mechanisms in plants, we explain why past measurements of D abundance yield unreliable paleo signals. Our data show that variation in D abundance among the C–H groups (isotopomer variation) of tree-ring cellulose is generally greater than variation in D abundance due to climatic influences. A comparison of the D isotopomer abundances of soluble sugars of annual plants and of trees, and of tree-ring cellulose shows that an “isotopomer pattern” of the C3 photosynthetic pathway is transmitted from soluble sugars to tree-ring cellulose. Differences in this pattern between oaks and conifers appear to be related to differences in metabolism. Furthermore, the patterns are modified by hydrogen isotope exchange between C–H groups and source water during cellulose synthesis. Based on these results, we propose a strategy to simultaneously reconstruct climate signals and signals related to tree physiology from D isotopomers of tree rings. Combination of climate signals and physiological signals may allow the detection of century-time-scale adaptations of trees to past environmental change, and help to forecast future adaptations.

  • 3.
    Augusti, Angela
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Betson, Tatiana R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Deuterium isotopomers record a CO2 response of plants in leaves and tree ringsManuscript (preprint) (Other (popular science, discussion, etc.))
  • 4.
    Augusti, Angela
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Betson, Tatiana R
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hydrogen exchange during cellulose synthesis distinguishes climatic and biochemical isotope fractionations in tree rings.2006In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 172, no 3, p. 490-499Article in journal (Refereed)
    Abstract [en]

    • The abundance of the hydrogen isotope deuterium (D) in tree rings is an attractive record of climate; however, use of this record has proved difficult so far, presumably because climatic and physiological influences on D abundance are difficult to distinguish.

    • Using D labelling, we created a D gradient in trees. Leaf soluble sugars of relatively low D abundance entered cellulose synthesis in stems containing strongly D-labelled water. We used nuclear magnetic resonance (NMR) spectroscopy to quantify D in the C-H groups of leaf glucose and of tree-ring cellulose.

    • Ratios of D abundances of individual C-H groups of leaf glucose depended only weakly on leaf D labelling, indicating that the D abundance pattern was determined by physiological influences. The D abundance pattern of tree-ring cellulose revealed C-H groups that exchanged strongly (C(2)-H) or weakly (C(6)-H2) with water during cellulose synthesis.

    • We propose that strongly exchanging C-H groups of tree-ring cellulose adopt a climate signal stemming from the D abundance of source water. C-H groups that exchange weakly retain their D abundance established in leaf glucose, which reflects physiological influences. Combining both types of groups may allow simultaneous reconstruction of climate and physiology from tree rings.

  • 5. Augusti, Angela
    et al.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The ins and outs of stable isotopes in plants.2007In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 174, no 3, p. 473-475Article in journal (Refereed)
  • 6.
    Betson, Tatiana R
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Augusti, Angela
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quantification of deuterium isotopomers of tree-ring cellulose using nuclear magnetic resonance.2006In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 24, p. 8406-8411Article in journal (Refereed)
    Abstract [en]

    Stable isotopes in tree rings are important tools for reconstruction of past climate. Deuterium (D) is of particular interest since it may contain climate signals and report on tree physiology. Measurements of the D/H ratio of tree-ring cellulose have proven difficult to interpret, presumably because the D/H ratio of the whole molecule blends the abundances of the seven D isotopomers of cellulose. Here we present a method to measure the abundance of the D isotopomers of tree-ring cellulose by nuclear magnetic resonance spectroscopy (NMR). The method transforms tree-ring cellulose into a glucose derivative that gives highly resolved, quantifiable deuterium NMR spectra. General guidelines for measurement of D isotopomers by NMR are described. The transformation was optimized for yield and did not alter the original D isotopomer abundances, thus, conserving the original signals recorded in wood cellulose. In the tree-ring samples tested, the abundances of D isotopomers varied by approximately ±10% (2% standard error). This large variability can only be caused by biochemistry processes and shows that more information is present in D isotopomer abundances, compared to the D/H ratio. Therefore, measurements of the D isotopomer distribution of tree rings may be used to obtain information on long-term adaptations to environmental changes and past climate change.

  • 7. Betson, Tatiana R.
    et al.
    Augusti, Angela
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quantifying deuterium isotopomers of cellulose using Nuclear Magnetic Resonance2006In: Analytical Chemistry, Vol. 78, no 24, p. 8406-8411Article in journal (Refereed)
  • 8. Chaintreau, Alain
    et al.
    Fieber, Wolfgang
    Sommer, Horst
    Gilbert, Alexis
    Yamada, Keita
    Yoshida, Naohiro
    Pagelot, Alain
    Moskau, Detlef
    Moreno, Aitor
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reniero, Fabiano
    Holland, Margaret
    Guillou, Claude
    Silvestre, Virginie
    Akoka, Serge
    Remaud, Gerald S
    Site-specific C-13 content by quantitative isotopic C-13 nuclear magnetic resonance spectrometry: a pilot inter-laboratory study2013In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 788, p. 108-113Article in journal (Refereed)
    Abstract [en]

    Isotopic C-13 NMR spectrometry, which is able to measure intra-molecular C-13 composition, is of emerging demand because of the new information provided by the C-13 site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic C-13 NMR as a routine tool. This paper describes the first collaborative study of intra-molecular C-13 composition by NMR. The main goals of the ring test were to establish intra-and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic C-13 NMR was then assessed on vanillin from three different origins associated with specific delta C-13(i) profiles. The standard deviation was, on average, between 0.9 and 1.2 parts per thousand for intra-variability. The highest standard deviation for inter-variability was 2.1%. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of delta C-13(i) in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.

    (C) 2013 Elsevier B.V. All rights reserved.

  • 9. Christodoulou, John
    et al.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Fucini, Paola
    Connell, Sean R
    Pertinhez, Thelma A
    Hanson, Charlotte L
    Redfield, Christina
    Nierhaus, Knud H
    Robinson, Carol V
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Dobson, Christopher M
    Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes.2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 101, no 30, p. 10949-54Article in journal (Refereed)
  • 10. Clementi, Emily A.
    et al.
    Wilhelm, Kristina R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Juergen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hakansson, Anders P.
    A Complex of Equine Lysozyme and Oleic Acid with Bactericidal Activity against Streptococcus pneumoniae2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. Article Number: UNSP e80649-Article in journal (Refereed)
    Abstract [en]

    HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET's bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA's bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments - an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes' bactericidal activities, while the proteins are required both to solubilize and/or present the lipid at the bacterial membrane and likely to confer other and separate functions during the bacterial death.

  • 11. Cormier, Marc-André
    et al.
    Werner, Roland A.
    Sauer, Peter E.
    Gröcke, Darren R.
    Leuenberger, Markus C.
    Wieloch, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kahmen, Ansgar
    2H-fractionations during the biosynthesis of carbohydrates and lipids imprint a metabolic signal on the δ2H values of plant organic compounds2018In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 218, no 2, p. 479-491Article in journal (Refereed)
    Abstract [en]

    Hydrogen (H) isotope ratio (δ2H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2H variability observed in plant compounds not being fully elucidated.

    We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ2H values of plant organic compounds.

    The experiments demonstrate that information recorded in the δ2H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where 2H‐fractionations occur in the biosynthesis of plant organic compounds and how these 2H‐fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems.

    Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.

  • 12. Cromsigt, J
    et al.
    van Buuren, B
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wijmenga, S
    Resonance assignment and structure determination for RNA.2001In: Methods Enzymol, ISSN 0076-6879, Vol. 338, p. 371-99Article in journal (Other academic)
  • 13. Cromsigt, Jenny
    et al.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Gustafsson, Tomas
    Kihlberg, Jan
    Wijmenga, Sybren
    Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides.2002In: Nucleic Acids Res, ISSN 1362-4962, Vol. 30, no 7, p. 1639-45Article in journal (Refereed)
  • 14. Drotz, Stina Harrysson
    et al.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nilsson, Mats B
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öquist, Mats G
    Both catabolic and anabolic heterotrophic microbial activity proceed in frozen soils2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 49, p. 21046-21051Article in journal (Refereed)
    Abstract [en]

    A large proportion of the global soil carbon pool is stored in soils of high-latitude ecosystems in which microbial processes and production of greenhouse gases proceed during the winter months. It has been suggested that microorganisms have limited ability to sequester substrates at temperatures around and below 0 °C and that a metabolic shift to dominance of catabolic processes occurs around these temperatures. However, there are contrary indications that anabolic processes can proceed, because microbial growth has been observed at far lower temperatures. Therefore, we investigated the utilization of the microbial substrate under unfrozen and frozen conditions in a boreal forest soil across a temperature range from -9 °C to +9 °C, by using gas chromatography-isotopic ratio mass spectrometry and (13)C magic-angle spinning NMR spectroscopy to determine microbial turnover and incorporation of (13)C-labeled glucose. Our results conclusively demonstrate that the soil microorganisms maintain both catabolic (CO(2) production) and anabolic (biomass synthesis) processes under frozen conditions and that no significant differences in carbon allocation from [(13)C]glucose into [(13)C]CO(2) and cell organic (13)C-compounds occurred between +9 °C and -4 °C. The only significant metabolic changes detected were increased fluidity of the cell membranes synthesized at frozen conditions and increased production of glycerol in the frozen samples. The finding that the processes in frozen soil are similar to those in unfrozen soil has important implications for our general understanding and conceptualization of soil carbon dynamics in high-latitude ecosystems.

  • 15.
    Drotz, Stina Harrysson
    et al.
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Öquist, Mats G
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Effects of soil organic matter composition on unfrozen water content and heterotrophic CO2 production of frozen soils2010In: Geochimica et Cosmochimica Acta, ISSN 0016-7037, E-ISSN 1872-9533, Vol. 74, no 8, p. 2281-90Article in journal (Refereed)
    Abstract [en]

    Several recent studies have highlighted the importance of soil organic matter (SOM) mineralization at high latitudes during winter for ecosystem carbon (C) balances, and the ability of the soil to retain unfrozen water at sub-zero temperatures has been shown to be a major determinant of C mineralization rates. Further, SOM is believed to strongly influence the liquid water contents in frozen surface layers of boreal forest soils and tundra, but the mechanisms and specific factors involved are currently unknown. Here we evaluate the effects of the chemical composition of SUM on the amount of unfrozen water, the pore size equivalents in which unfrozen water can exist, and the microbial heterotrophic activity at sub-zero temperatures in boreal forest soils. To do this, we have characterized the chemical composition of SUM in forest soil samples (surface O-horizons) using solid state CP-MAS (cross polarization magic angle spinning) NMR spectroscopy. The acquired information was then used to elucidate the extent to which different fractions of SUM can explain the observed variations in unfrozen water content, pore size equivalents, and biogenic CO2 production rates in the examined soil samples under frozen conditions (-4 degrees C). The data evaluation was done by the use of principal component analysis (PCA) and projections to latent structures by means of partial least square (PLS). We conclude that aromatic, O-aromatic, methoxy/N-alkyl and alkyl C are the major SOM components affecting frozen boreal forest soil's ability to retain unfrozen water and sustain heterotrophic activity (95% confidence level). Our results reveal that solid carbohydrates have a significant negative impact (95% confidence level) on CO2 production in frozen boreal spruce forest soils, in contrast to the positive effects of carbohydrate polymers during unfrozen conditions. We conclude that the hierarchy of environmental factors controlling SOM mineralization changes as soils freeze. The effect of SUM composition on pore size distribution and unfrozen water content has a superior influence on SUM mineralization and hence on heterotrophic CO2 production of frozen soils. (C) 2010 Elsevier Ltd. All rights reserved.

  • 16. Duchardt, Elke
    et al.
    Nilsson, Lennart
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study.2008In: Nucleic acids research, ISSN 1362-4962, Vol. 36, no 12, p. 4211-9Article in journal (Refereed)
    Abstract [en]

    Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.

  • 17.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Augusti, Angela
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Betson, Tatiana R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B.
    Marshall, John D.
    Schleucher, Juergen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C-3 plants over the 20th century2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 51, p. 15585-15590Article in journal (Refereed)
    Abstract [en]

    Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C-3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C-3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C-3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the similar to 100-ppm CO2 increase between similar to 1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.

  • 18. Ehlers, Ina
    et al.
    Augusti, Angela
    Köhler, Iris
    Wieloch, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zuidema, Pieter
    Robertson, Iain
    Nilsson, Mats
    Marshall, John
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Detecting plant-climate interactions over decades-millennia using NMR isotopomer analysis2016In: Geophysical Research Abstracts, 2016, Vol. 18, article id EGU2016-9141-2Conference paper (Refereed)
  • 19.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Betson, Tatiana R.
    Vetter, Walter
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis: The Persistent Organic Pollutant DDT2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, p. e110648-Article in journal (Refereed)
    Abstract [en]

    The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight againstmalaria, although DDT and related compounds  pose toxicological  hazards. Technical DDT contains the dichloro congenerDDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene)   as by-product, but  DDD is also formed by  reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical  DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis.  In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess  DDT turnover in the environment. Because  intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of smallbioactive molecules in chemistry, physiology and environmental science.

  • 20.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Köhler, Iris
    Wieloch, Thomas
    Vlam, Mart
    van der Sleen, Peter
    Groenendijk, Peter
    Grabner, Michael
    Seim, Andrea
    Allen, Kathryn
    Wei, Liang
    Robertson, Iain
    Marshall, John
    Zuidema, Pieter A.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Limited suppression of photorespiration by 20th century atmospheric CO2 increase in trees worldwideManuscript (preprint) (Other academic)
    Abstract [en]

    Forests are a key component of the global carbon and hydrological cycle and forest responses to  environmental  drivers  create  important  feedbacks  to  these  cycles.  Photosynthetic efficiency of most forest tree species is strongly limited by photorespiration, a side reaction using O2 instead of CO2 as substrate, leading to a carbon loss for the plant. Photorespiration occurs in all trees and is reduced under elevated CO2 concentrations and increased under elevated temperature. Because the CO2 concentration of the atmosphere has increased in past decades, long-lived trees may have benefited from reduced photorespiration, but the temperature increase would have been a compensating detriment; but direct quantification of long-term changes in metabolic fluxes is lacking. Realistic forecasting of responses of trees and forests to future CO2 and temperature demands quantifying the reduction of photorespiration.  In  twelve  tree  species  from  five  continents,  we  observe  that photorespiration has been reduced by the CO2 increase during the past century, but for most the reduction is smaller than predicted from plant responses in CO2 alone. Comparison with data from a combined CO2 and temperature manipulation experiment shows that the reduced response can be explained by increases in leaf temperatures, which might result directly from increased  air  temperatures  or  indirectly  from  reduced  transpirative  cooling.  These  data suggest that global warming has already inhibited plant fertilization by increasing CO2, and that biomass increases may have been smaller than deduced from measurements of the heavy carbon isotope 13C. Observation of this centennial metabolic shift in tree physiology worldwide provides new insights into forest-climate feedbacks and can be used to improve coupled climate-vegetation models.

  • 21. Ehlers, Ina
    et al.
    Wieloch, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Groenendijk, Peter
    Vlam, Mart
    van der Sleen, Peter
    Zuidema, Pieter A.
    Robertson, Iain
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enhanced photosynthetic efficiency in trees world-wide by rising atmospheric CO2 levels2014In: Geophysical Research Abstracts, 2014, Vol. 16, article id EGU2014-12587-1Conference paper (Refereed)
  • 22.
    Erhagen, Björn
    et al.
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Öquist, Mats
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haei, Mahsa
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Ilstedt, Ulrik
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Temperature response of litter and soil organic matter decomposition is determined by chemical composition of organic material2013In: Global Change Biology, ISSN 1354-1013, E-ISSN 1365-2486, Vol. 19, no 12, p. 3858-3871Article in journal (Refereed)
    Abstract [en]

    The global soil carbon pool is approximately three times larger than the contemporary atmospheric pool, therefore even minor changes to its integrity may have major implications for atmospheric CO2 concentrations. While theory predicts that the chemical composition of organic matter should constitute a master control on the temperature response of its decomposition, this relationship has not yet been fully demonstrated. We used laboratory incubations of forest soil organic matter (SOM) and fresh litter material together with NMR spectroscopy to make this connection between organic chemical composition and temperature sensitivity of decomposition. Temperature response of decomposition in both fresh litter and SOM was directly related to the chemical composition of the constituent organic matter, explaining 90% and 70% of the variance in Q10 in litter and SOM respectively. The Q10 of litter decreased with increasing proportions of aromatic and O-aromatic compounds, and increased with increased contents of alkyl- and O-alkyl carbons. In contrast, in SOM, decomposition was affected only by carbonyl compounds. To reveal why a certain group of organic chemical compounds affected the temperature sensitivity of organic matter decomposition in litter and SOM, a more detailed characterisation of the (13) C aromatic region using Heteronuclear Single Quantum Coherence (HSQC) was conducted. The results revealed considerable differences in the aromatic region between litter and SOM. This suggests that the correlation between chemical composition of organic matter and the temperature response of decomposition differed between litter and SOM. The temperature response of soil decomposition processes can thus be described by the chemical composition of its constituent organic matter, this paves the way for improved ecosystem modelling of biosphere feedbacks under a changing climate.

  • 23.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Cromsigt, Jenny
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.2002In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 19, no 4, p. 627-636Article in journal (Other academic)
    Abstract [en]

    Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.

  • 24.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kidd-Ljunggren, K
    Wijmenga, S
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mobile nucleotides mediate binding of HBV reverse transcriptase to its RNA targetManuscript (preprint) (Other academic)
  • 25.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Petersen, Michael
    Girard, Frederic
    Zdunek, Janusz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kidd-Ljunggren, Karin
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wijmenga, Sybren
    Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 16, p. 4449-4457Article in journal (Refereed)
    Abstract [en]

    Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

  • 26.
    Flodell, Sara
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Cromsigt, Jenny
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Ippel, Hans
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.2002In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 21, p. 4803-4811Article in journal (Refereed)
    Abstract [en]

    Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.

  • 27.
    Greule, Markus
    et al.
    Max Planck Inst for Chemistry.
    Tumino, Luisa Dana
    Inst Lebensmittelchem, JW Goethe Univ Frankfurt.
    Kronewald, Tatjana
    Hener, Uwe
    Inst Lebensmittelchem, JW Goethe Univ Frankfurt.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mosandl, Armin
    Max Planck Inst for Chemistry.
    Keppler, Frank
    Max Planck Inst for Chemistry.
    Improved rapid authentication of vanillin using δ13C and δ2H values2010In: European Food Research and Technology, ISSN 1438-2377, E-ISSN 1438-2385, Vol. 231, no 6, p. 933-941Article in journal (Refereed)
    Abstract [en]

    Vanilla still remains one of the most important and widely used flavours in the food industry and is also extensively employed by fragrance and pharmaceutical manufacturing companies. Natural vanilla flavour, extracted from the pods of the tropic orchid vanilla, is considerably more expensive than synthetic vanillin. The disparity of prices between natural vanillin and that derived from other sources has given rise to many cases of fraudulent adulteration, and for more than 30 years, strenuous efforts have been made to authenticate sources of vanillin. Stable isotope analysis is one of the most powerful analytical tools to distinguish between natural vanillin and that originating from other sources. Recently, a rapid and precise method for analysis of both δ13C and δ2H values of plant methoxyl groups has been published. Here, we report an application of the method for the control of authenticity of vanillin. Carbon and hydrogen stable isotope values of the vanillin molecule and vanillin methoxyl groups of vanillin samples of different origins including authentic and synthetic samples were measured. The results clearly show that use of this approach provides a rapid and reliable authenticity assessment of vanillin. The technique used for these studies is robust and rapid, involves minimum sample preparation and requires only a small amount of vanillin sample, usually 1 mg for stable carbon and 4 mg for stable hydrogen analysis.

  • 28. Harrysson Drotz, Stina
    et al.
    Tilston, Emma L
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Contributions of matric and osmotic potentials to the unfrozen water content of frozen soils2009In: Geoderma, ISSN 0016-7061, E-ISSN 1872-6259, Vol. 148, no 3-4, p. 392-8Article in journal (Refereed)
    Abstract [en]

    Recent reports show that biogeochemical processes continue when the soil is frozen, but are limited by water availability. However, there is little knowledge about the interactive effects of soil and environmental variables on amounts of unfrozen water in frozen soils. The aims of this study were to determine the contributions of matric and osmotic potentials to the unfrozen water content of frozen soil. We determined the effects of matric and osmotic potential on unfrozen water contents of frozen mineral soil fractions (ranging from coarse sand to fine silt) at − 7 °C, and estimated the contributions of these potentials to liquid water contents in samples from organic surface layers of boreal soils frozen at − 4 °C. In the mineral soil fractions the unfrozen water contents appeared to be governed solely by the osmotic potential, but in the humus layers of the sampled boreal soils both the osmotic and matric potentials control unfrozen water content, with osmotic potential contributing 20 to 69% of the total water potential. We also determined pore size equivalents, where unfrozen water resides at − 4 °C, and found a strong correlation between these equivalents and microbial CO2 production. The larger the pores in which the unfrozen water is found the larger the microbial activity that can be sustained. The osmotic potential may therefore be a key determinant of unfrozen water and carbon dynamics in frozen soil.

  • 29.
    Hägglund, Gunnar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morgenstern, Ralf
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    1915-Pos 195 Pt Nmr: Interactions Of The Cancer Drug Cis-platin With Membranes And Mgst1, A Integral Membrane Detoxification Protein2008In: Biophysical Journal, 2008, p. 1915-Conference paper (Other academic)
    Abstract [en]

    The microsomal Glutathione-transferase (MGST1) is an integral membrane protein, which catalyses the conjugation of glutathione (tripeptide GSH) with xenobiotics; a process essential for cells to remove and detoxify e. g. carcinogens. While this glutathione system plays an essential role in healthy cell survival, glutathione has been shown to have a pivotal role in the development of acquired drug resistance. It prevents successful chemotherapies against a range of cancer types, therapies often based on cisplatin based drugs. These Pt compounds are initially quite effective, they become non-effective e.g. during the treatment of prostate cancer (very common 10000 new cases/a in Sweden) which progresses into a non-curable form during therapy. To understand the molecular mechanism behind the activity of Pt drugs and their inhibition by the human defense system, we use an solid state NMR approach (complemented by liquid NMR) to elucidate for cis-platin (diamino-dichloroplatinat II):

    conversion of cis-platin complex into an diamino-diaqua-complex, essential for its membrane passage into the cell interior.

    lipid membrane - drug interactions: binding to cell membrane surface, solubility and membrane transport.

    Pt drug binding to MGST1 enzyme, followed by glutathione conjugation into more water soluble compounds.

  • 30. Ippel, H
    et al.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Behravan, G
    Zdunek, J
    Lundqvist, M
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lycksell, P O
    Wijmenga, S
    The solution structure of the homeodomain of the rat insulin-gene enhancer protein isl-1. Comparison with other homeodomains.1999In: J Mol Biol, ISSN 0022-2836, Vol. 288, no 4, p. 689-703Article in journal (Refereed)
  • 31. Ippel, J H
    et al.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Behravan, G
    Lundqvist, M
    Lycksell, P O
    Schleucher, Jurgen
    Medical Biosciences.
    Zdunek, J
    Wijmenga, S S
    1H, 13C and 15N assignment of the Isl-1 homeodomain.1998In: J Biomol NMR, ISSN 0925-2738, Vol. 12, no 2, p. 357-9Article in journal (Refereed)
  • 32. Ippel, Johannes H
    et al.
    Olofsson, Anders
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Lundgren, Erik
    Wijmenga, Sybren S
    Probing solvent accessibility of amyloid fibrils by solution NMR spectroscopy.2002In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 99, no 13, p. 8648-53Article in journal (Refereed)
  • 33. Kaffarnik, Stefanie
    et al.
    Ehlers, Ina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vetter, Walter
    Two-Dimensional P-31,H-1 NMR Spectroscopic Profiling of Phospholipids in Cheese and Fish2013In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 61, no 29, p. 7061-7069Article in journal (Refereed)
    Abstract [en]

    Phospholipids (PLs) comprise an important lipid class in food because of their technological use as emulsifiers and their nutritional value. This study used one-dimensional P-31 NMR and two-dimensional (2D) P-31,H-1 COSY NMR spectroscopy for the determination of the PL composition of cheese and fish after liquid liquid enrichment. This extraction step enabled the identification of 10 PLs in cheese and 9 PLs in fish by 2D P-31,H-1 NMR. Variations in the P-31 shifts indicated differences in the fatty acids attached to the individual PLs. The total PL content in cheese fat and fish oil ranged from 0.3 to 0.4% and from 5 to 12%, respectively. Phosphatidylcholine was the most prominent PL in both matrices (up to 6596). Minor PLs (limit of detection = 4 nmol, i.e. 500 mu L of an 8 mu M solution) were identified in forms of phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol. Specific cross couplings and H-1 fine structures in the 2D P-31,H-1 NMR spectra proved to be valuable for the assignment and verification of known and uncommon PLs in the samples.

  • 34.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Martinez, Gary
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wijmenga, Sybren S
    Detection of nano-second internal motion and determination of overall tumbling times independent of the time scale of internal motion in proteins from NMR relaxation data.2003In: J Biomol NMR, ISSN 0925-2738, Vol. 27, no 4, p. 291-312Article in journal (Refereed)
  • 35.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Saarikettu, Juha
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sveshnikova, Natalia
    Zdunek, Janusz
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Grundström, Thomas
    Wijmenga, Sybren
    A third principle of protein:protein recognition: the calmodulin dimer interaction with basic-helix-loop-helix transcription factorsManuscript (Other academic)
  • 36.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Onions, J
    Hermann, S
    Grundström, T
    Wijmenga, S S
    A novel target recognition revealed by calmodulin in complex with the basic helix--loop--helix transcription factor SEF2-1/E2-2.2001In: Protein Sci, ISSN 0961-8368, Vol. 10, no 1, p. 169-86Article in journal (Other academic)
  • 37.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Onions, Jacqueline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hermann, Stefan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wijmenga, Sybren S
    Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix-loop-helix transcription factor SEF2-1/E2-2: a highly dynamic complex.2005In: Biophys J, ISSN 0006-3495, Vol. 89, no 2, p. 1214-26Article in journal (Refereed)
    Abstract [en]

    Calmodulin (CaM) interacts specifically as a dimer with some dimeric basic-Helix-Loop-Helix (bHLH) transcription factors via a novel high affinity binding mode. Here we report a study of the backbone dynamics by (15)N-spin relaxation on the CaM dimer in complex with a dimeric peptide that mimics the CaM binding region of the bHLH transcription factor SEF2-1. The relaxation data were measured at multiple magnetic fields, and analyzed in a model-free manner using in-house written software designed to detect nanosecond internal motion. Besides picosecond motions, all residues also experience internal motion with an effective correlation time of approximately 2.5 ns with squared order parameter (S(2)) of approximately 0.75. Hydrodynamic calculations suggest that this can be attributed to motions of the N- and C-terminal domains of the CaM dimer in the complex. Moreover, residues with significant exchange broadening are found. They are clustered in the CaM:SEF2-1mp binding interface, the CaM:CaM dimer interface, and in the flexible helix connecting the CaM N- and C-terminal domains, and have similar exchange times (approximately 50 micros), suggesting a cooperative mechanism probably caused by protein:protein interactions. The dynamic features presented here support the conclusion that the conformationally heterogeneous bHLH mimicking peptide trapped inside the CaM dimer exchanges between different binding sites on both nanosecond and microsecond timescales. Nature has thus found a way to specifically recognize a relatively ill-fitting target. This novel mode of target-specific binding, which neither belongs to lock-and-key nor induced-fit binding, is characterized by dimerization and continuous exchange between multiple flexible binding alternatives.

  • 38.
    Larsson, Göran
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wijmenga, Sybren S
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A high-resolution HCANH experiment with enhanced sensitivity via multiple quantum line narrowing1999In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 14, no 2, p. 169-174Article in journal (Refereed)
    Abstract [en]

    We report a 3D constant-time HCANH experiment (CTSL-HCANH) that uses the slower relaxation of multiple-quantum coherence to increase sensitivity and provides high C(α) resolution. In this experiment the H(α) of the (H(α), C(α)) multiple quanta are selectively spin locked, so that H(α) chemical shift evolution and (1) H-(1)H J-dephasing become ineffective during the relatively long delay needed for C(α) to N coherence transfer. As compared to an HCANH experiment that uses C(α) single-quantum coherence, an average enhancement of 20% was observed on calmodulin in complex with the binding domain of the transcription factor SEF2-1. Compared to CBCANH the signal intensity is approximately twice as good. The favorable relaxation properties of multiple quanta, together with the outstanding C(α) resolution, make the experiment a very good complement to CBCANH and CBCA(CO)NH for sequential assignment of larger proteins for which deuteration is not yet necessary.

  • 39.
    Nielsen, Søren B
    et al.
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    Wilhelm, Kristina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vad, Brian
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Otzen, Daniel
    Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
    The interaction of equine lysozyme: oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 398, no 2, p. 351-361Article in journal (Refereed)
    Abstract [en]

    The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOA) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appears to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells (HAMLET). Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation (QCM-D) data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and "soft" lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with BSA, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA which shifts towards free OA as ELOA is progressively diluted indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA toward a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein. Abbreviations QCM-D, Quartz crystal microbalance with dissipation; CD, Circular dichroism; EL, equine lysozyme; ELOA, EL complex with oleic acid; OA, oleic acid, CSLM, Confocal scanning laser microscopy, Df, dissipation-frequency.

  • 40.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Petzold, Katja
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Haas, Rainer
    Max-von-Pettenkofer-Institute of Hygiene and Medical Microbiology, Dept of Bacteriology, Munich, Germany.
    Backert, Steffen
    School of Biomolecular and Biomedical Sciences, University College Dublin, Ireland.
    Nyunt Wai, Sun
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Characterization of Helicobacter pylori vesicles and their cognate properties for intimate host interactionsManuscript (preprint) (Other academic)
  • 41.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallström, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Petzold, Katja
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tegtmeyer, Nicole
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Haas, Rainer
    Backert, Steffen
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Biochemical and functional characterization of Helicobacter pylori vesicles2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 6, p. 1539-1555Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigor of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and NMR. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.

  • 42.
    Petzold, Katja
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Duchardt, Elke
    Flodell, Sara
    Larsson, Göran
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Kidd-Ljunggren, Karin
    Wijmenga, Sybren
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 20, p. 6854-6861Article in journal (Refereed)
  • 43.
    Petzold, Katja
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Annelie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jürgen, Schleucher
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Semiconstant-Time P,H-COSY NMR: Analysis of Complex Mixtures of Phospholipids Originating from Helicobacter pylori2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 40, p. 14150-1Article in journal (Refereed)
    Abstract [en]

    Lipids play a central role in numerous biological events, ranging from normal physiological processes to host−pathogen interactions. The proposed semiconstant-time 31P,1H−COSY NMR experiment provides identification of known and structural characterization of unknown phospholipids in complex membrane extracts with high sensitivity, based on the combination of their 1H and 31P chemical shifts and coupling patterns. Furthermore, the spectra allow quantification of phospholipid composition. Analysis of the phospholipid composition of Helicobacter pylori, the causative agent of peptic ulcer disease, showed the presence of uncommon phospholipids. This novel NMR approach allows the study of changes in membrane composition in response to biological stimuli and opens up the possibility of identifying soluble phosphorus species in a number of research fields.

  • 44. Qian, H
    et al.
    Rogers, M S
    Schleucher, Jurgen
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Edlund, U
    Strehler, E E
    Sethson, I
    Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy.1998In: Protein Sci, ISSN 0961-8368, Vol. 7, no 11, p. 2421-30Article in journal (Refereed)
  • 45.
    Rundqvist, Louise
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tengel, Tobias
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zdunek, Janusz
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Alcocer, Marcos J C
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Solution structure, copper binding and backbone dynamics of recombinant Ber e 1: the major allergen from brazil nut2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, p. e46435-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15)N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+) in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+) ions. This Cu(2+) binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+) can in turn affect the interaction that is essential to provoke an allergenic response.

  • 46.
    Sattler, Michael
    et al.
    EMBL, Heidelberg, Germany.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Griesinger, Christian
    Univ Frankfurt, Inst Organ Chem, Frankfurt, Germany.
    Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients1999In: Progress in nuclear magnetic resonance spectroscopy, ISSN 0079-6565, E-ISSN 1873-3301, Vol. 34, no 2, p. 93-158Article in journal (Refereed)
  • 47.
    Schleucher, Jurgen
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sattler, M.
    Griesinger, C.
    Coherence Selection by Gradients without Signal Attenuation: Application to the 3-Dimensional Hnco Experiment1993In: Angewandte Chemie-International Edition in English, Vol. 32, no 10, p. 1489-1491Article in journal (Refereed)
  • 48.
    Schleucher, Jurgen
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Vanderveer,
    Sharkey,
    Export of carbon from chloroplasts at night1998In: Plant Physiol, ISSN 0032-0889, Vol. 118, no 4, p. 1439-45Article in journal (Refereed)
  • 49.
    Schleucher, Jurgen
    et al.