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  • 1.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundgren, Camilla A. K.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magnúsdóttir, Auður
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martinez Molina, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mycobacterium tuberculosis Very-Long-Chain Fatty Acyl-CoA Synthetase: Structural Basis for Housing Lipid Substrates Longer than the Enzyme2012In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 20, no 6, p. 1062-1070Article in journal (Refereed)
    Abstract [en]

    The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.

  • 2.
    Bakali, Amin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Herman, Maria Dolores
    Johnson, Kenneth A.
    Kelly, Amélie A.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hallberg, B. M.
    Nordlund, Pär
    Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, p. 19644-19652Article in journal (Refereed)
    Abstract [en]

    The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

  • 3.
    Eriksson, Hanna M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Georgiev, Alexander
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing proteinManuscript (preprint) (Other academic)
    Abstract [en]

    Escherichia coli has a limited capacity to overexpress integral membrane proteins to amounts needed for structural studies. This is usually attributed to the limited capacity of the Sec transport machinery, shortage of accessory chaperons, sub-optimal codon usage, potentially “wrong” lipids, and lack of membrane space for the new proteins. A foreign, monotopic lipid glycosyltransferase was recently shown to induce the formation of extensive amounts of intracellular vesicles in E. coli. We show here that such vesicles can improve the expressed levels up to 3-4 times for a substantial fraction of integral membrane proteins tested. These had 2 to 12 transmembrane helices, and all had a C-terminally fused GFP reporter. Strongly overexpressed proteins yielded intensely green vesicles, of slightly lower buoyant density than the inner membranes. Most proteins could be detected in the vesicles. Multivariate sequence analyses indicated a correlation between sequence property features and expression levels, and factors analyzed involved protein mass, transmembrane segments, inside/outside loops, etc. It is concluded that this vesicular system can yield substantial improvements in expression levels, by creation of extra membranes and lateral space in E. coli.

  • 4.
    Eriksson, Hanna M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Persson, Karina
    Umeå Universitet.
    Zhang, Shuguang
    Massachusetts Institute of Technology.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High-yield expression and purification of a monotopic membrane glycosyltransferase2009In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 2, p. 143-148Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-Dodecyl-β-D-maltoside (DDM) (~1× critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.

  • 5.
    Eriksson, Hanna M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wessman, Per
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Edwards, Katarina
    Wieslander, Ake
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 49, p. 33904-33914Article in journal (Refereed)
    Abstract [en]

    The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

  • 6.
    Ge, Changrong
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Georgiev, Alexander
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhman, Anders
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kelly, Amélie A.
    Tryptophan residues promote membrane association for a plant lipid glycosyltransferase involved in phosphate stress2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 8, p. 6669-6684Article in journal (Refereed)
    Abstract [en]

    Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

  • 7.
    Ge, Changrong
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Raussens, Vincent
    Ruysschaert, Jean-Marie
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modulation of Escherichia coli Cell Membrane by a Monotopic Lipid Glycosyltransferase - an Exploration of Potential MechanismsManuscript (preprint) (Other academic)
    Abstract [en]

    Intracellular vesicles are abundant in eukaryotic cells but are rare in Gram-negative bacterium Escherichia coli. Strongly overexpression of a monotopic glycolipid-synthesizing enzyme could induce massive formation of “foreign” vesicles in the cytoplasm. Here we investigate how this membrane-associated enzyme is able to bend and deform the plasma membrane. Limited proteolysis combined with ESI-MS suggested interface binding is mediated through both its two Rossmann fold topological domains. Detailed subcellular localization and liposome binding assay indicates different interface anchoring regions in the protein, and anionic lipid seems to influence the binding properties of the anchoring segments. Genetic engineering of a known membrane-bound segment to explore its vesiculation potentials led to the identification of important catalytic residues (regions). Flow cytometry and infrared spectroscopy were also performed on bacterial cells to get more insight into the cellular morphology and internal complexity. The linking region between two domains was demonstrated to be crucial for both catalytic function and vesiculation capacity of the enzyme. Based on our findings, we propose, that scaffold-like structural feature of this enzyme is most likey one of key elements contributing to vesiculation.        

  • 8.
    Georgiev, Alexander
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Basic clusters and amphipathic helices contribute to interactions of Myr1/Syh1 with membrane phospholipidsManuscript (preprint) (Other academic)
    Abstract [en]

    The ability to associate transiently with membrane bilayers is an important property of many protein regulators of membrane trafficking, lipid transfer proteins, or signaling modules. Membrane association is also a property of Myr1/Syh1, a soluble GYF domain protein from Saccharomyces cerevisiae, previously reported to rescue the temperature sensitive growth of ypt6 and ric1 null strains. Here, we further demonstrate that MYR1 also rescued the vacuole fragmentation phenotype of the ypt6 and ric1 mutants. The mechanism behind these genetic interactions is likely linked to the capacity of the Myr1/Syh1 protein to associate with phospholipid membranes. In order to elucidate further the nature of the interactions with vesicular traffic, we studied protein-protein and protein-phospholipid association of isolated domains from Myr1/Syh1. Using a two-hybrid assay, we confirmed the capacity of Myr1/Syh1 to self-associate in vivo. We measured in vitro the affinity of recombinant Myr1/Syh1 domains fused to GFP for liposomes reconstituted from synthetic and natural yeast lipids by sedimentation techniques. The herewith established affinities of Myr1/Syh1 to specific lipids, combined with evidence for its interactions with membrane traffic and protein synthesis, provide support for a possible function of Myr1/Syh1 as a regulator sensing membrane composition along the vesicular pathways.

  • 9.
    Georgiev, Alexander
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöström, Michael
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Binding specificities of the GYF domains from two Saccharomyces cerevisiae paralogs.2007In: Protein Eng Des Sel, ISSN 1741-0126, Vol. 20, no 9, p. 443-52Article in journal (Refereed)
    Abstract [en]

    We have used multivariate statistics and z-scales to represent peptide sequences

    in a PLS-QSAR model of previously studied binding affinities [Kofler,M.,

    Motzny,K. and Freund,C. (2005b) Mol. Cell. Proteomics, 4, 1797-1811.] of two GYF

    domains to an array of immobilized synthetic peptides. As a result, we

    established structural determinants of the binding specificities of the two

    proteins. Our model was used to define new sets of yeast proteins potentially

    interacting with Syh1 (YPL105C) and Smy2 (YBR172C). These sets were subsequently

    examined for co-occurrence of Gene Ontology terms, leading to suggest a group of

    likely interacting proteins with a common function in mRNA catabolism. Finally,

    subcellular localization of a GFP-fused Syh1 and Smy2 reinforced the possibility

    that these proteins reside in cytoplasmic sites of mRNA degradation, thereby

    providing experimental confirmation to the predictions from the model.

  • 10.
    Keller, Rebecca
    et al.
    Physiologie der Mikroorganismen, Humboldt Universität.
    Stenberg Bruzell, Filippa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Burstedt, Malin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hunke, Sabine
    Physiologie der Mikroorganismen, Humboldt Universität.
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The CpxA sensor and other cell envelope proteins respond to bilayer stress in Escherichia coliManuscript (preprint) (Other academic)
  • 11.
    Lagerstedt, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Voss, JC
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Persson, B L
    Structural modeling of dual-affinity purified Pho84 phosphate transporter2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 578, no 3, p. 262-268Article in journal (Refereed)
    Abstract [en]

    The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.

  • 12.
    Lind, Jesper
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rämö, Tuulia
    Rosén Klement, Maria L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bárány-Wallje, Elsa
    Epand, Richard M.
    Epand, Raquel F.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, p. 5664-5677Article in journal (Refereed)
    Abstract [en]

    In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

  • 13.
    Pisareva, Tatiana
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kwon, Joseph
    Oh, Jihyun
    Kim, Young H.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Choi, Jong-Soon
    Norling, Birgitta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Model for Membrane Organization and Protein Sorting in the Cyanobacterium Synechocystis sp. PCC 6803 Inferred from Proteomics and Multivariate Sequence Analyses2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 8, p. 3617-3631Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.

  • 14. Rajalahti, Tarja
    et al.
    Huang, Fang
    Klement, Maria Rosén
    Pisareva, Tatiana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Edman, Maria
    Sjöström, Michael
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norling, Birgitta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteins in different Synechocystis compartments have distinguishing N-terminal features: a combined proteomics and multivariate sequence analysis2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 7, p. 2420-2434Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria have a cell envelope consisting of a plasma membrane, a periplasmic space with a peptidoglycan layer, and an outer membrane. A third, separate membrane system, the intracellular thylakoid membranes, is the site for both photosynthesis and respiration. All membranes and luminal spaces have unique protein compositions, which impose an intriguing mechanism for protein sorting of extracytoplasmic proteins due to single sets of translocation protein genes. It is shown here by multivariate sequence analyses of many experimentally identified proteins in Synechocystis, that proteins routed for the different extracytosolic compartments have correspondingly different physicochemical properties in their signal peptide and mature N-terminal segments. The full-length mature sequences contain less significant information. From these multivariate, N-terminal property-profile models for proteins with single experimental localization, proteins with ambiguous localization could, to a large extent, be predicted to a defined compartment. The sequence properties involve amino acids varying especially in volume and polarizability and at certain positions in the sequence segments, in a manner typical for the various compartment classes. Potential means of the cell to recognize the property features are discussed, involving the translocation channels and two Type I signal peptidases with different cellular localization, and charge features at their membrane interfaces.

  • 15. Rosén Klement, Maria L.
    et al.
    Öjemyr, Linda
    Tagscherer, Katrin E.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Wieslander, Åke
    Department of Biochemistry and Biophysics.
    A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response2007In: Molecular Microbiology, ISSN 1365-2958, Vol. 65, no 6, p. 1444-1457Article in journal (Refereed)
  • 16.
    Szpryngiel, Scarlett
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lakovleva, Irina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Georgiev, Alexander
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lind, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 21, p. 4451-4466Article in journal (Refereed)
    Abstract [en]

    Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

  • 17. Tjellström, Henrik
    et al.
    Hellgren, Lars I.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sandelius, Anna Stina
    Lipid asymmetry in plant plasma membranes: phosphate deficiency-induced phospholipid replacement is restricted to the cytosolic leaflet2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 4, p. 1128-1138Article in journal (Refereed)
    Abstract [en]

    As in other eukaryotes, plant plasma membranes contain sphingolipids, phospholipids, and free sterols. In addition, plant plasma membranes also contain sterol derivatives and usually <5 mol% of a galactolipid, digalactosyldiacylglycerol (DGDG). We earlier reported that compared to fully fertilized oats (Avena sativa), oats cultivated without phosphate replaced up to 70 mol% of the root plasma membrane phospholipids with DGDG. Here, we investigated the implications of a high DGDG content on membrane properties. The phospholipid-to-DGDG replacement almost exclusively occurred in the cytosolic leaflet, where DGDG constituted up to one-third of the lipids. In the apoplastic (exoplasmic) leaflet, as well as in rafts, phospholipids were not replaced by DGDG, but by acylated sterol glycosides. Liposome studies revealed that the chain ordering in free sterol/phospholipid mixtures clearly decreased when > 5mol% DGDG was included. As both the apoplastic plasma membrane leaflet (probably the major water permeability barrier) and rafts both contain only trace amounts of DGDG, we conclude that this lipid class is not compatible with membrane functions requiring a high degree of lipid order. By not replacing phospholipids site specifically with DGDG, negative functional effects of this lipid in the plasma membrane are avoided.

  • 18. Wikström, Malin
    et al.
    Kelly, Amélie A
    Georgiev, Alexander
    Eriksson, Hanna M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klement, Maria Rosén
    Bogdanov, Mikhail
    Dowhan, William
    Wieslander, Ake
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.2009In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 2, p. 954-65Article in journal (Refereed)
    Abstract [en]

    Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

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