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  • 1.
    Aisenbrey, Christopher
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bechinger, Burkhard
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular Crowding at Membrane Interfaces: Adsorption and Alignment of Membrane Peptides2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, p. 376-385Article in journal (Refereed)
    Abstract [en]

    Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH4, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.

    High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.

  • 2.
    Aisenbrey, Christopher
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Borowik, Tomasz
    Byström, Roberth
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bokvist, Marcus
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrick
    Umeå University, Faculty of Science and Technology, Chemistry.
    Misiak, Hanna
    Sani, Marc-Antoine
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    How is protein aggregation in amyloidogenic diseases modulated by biological membranes?2008In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, European Biophysics Journal, ISSN 1432-1017 (Online), Vol. 37, no 3, p. 247-55Article in journal (Refereed)
    Abstract [en]

    The fate of proteins with amyloidogenic properties depends critically on their immediate biochemical environment. However, the role of biological interfaces such as membrane surfaces, as promoters of pathological aggregation of amyloidogenic proteins, is rarely studied and only established for the amyloid-β protein (Aβ) involved in Alzheimer’s disease, and α-synuclein in Parkinsonism. The occurrence of binding and misfolding of these proteins on membrane surfaces, is poorly understood, not at least due to the two-dimensional character of this event. Clearly, the nature of the folding pathway for Aβ protein adsorbed upon two-dimensional aggregation templates, must be fundamentally different from the three-dimensional situation in solution. Here, we summarize the current research and focus on the function of membrane interfaces as aggregation templates for amyloidogenic proteins (and even prionic ones). One major aspect will be the relationship between membrane properties and protein association and the consequences for amyloidogenic products. The other focus will be on a general understanding of protein folding pathways on two-dimensional templates on a molecular level. Finally, we will demonstrate the potential importance of membrane-mediated aggregation for non-amphiphatic soluble amyloidogenic proteins, by using the SOD1 protein involved in the amyotrophic lateral sclerosis syndrome.

  • 3.
    Aisenbrey, Christopher
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Byström, Roberth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oliveberg, Mikael
    Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    SOD1 associates to membranes in its folded apo-stateManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease accompanied by misfolding and intracellular deposition of superoxide dismutase 1 (SOD1). Although the molecular details behind this misfolding process are yet poorly understood, increasing evidence suggest that SOD1 is most susceptible to misfolding in its metal-free and relatively unstable apo-state. Here, we addressed the question, if misfolding and aggregation of SOD1 involves erroneous interactions with membranes as has been implicated for the Aβ peptide in Alzheimers disease. To examine this possibility we subjected various apo SOD1 variants to the presence of different membrane systems. The results reveal that wild type apoSOD1 but to less extent destabilized ALS mutations interact with charged vesicles under physiologically relevant conditions, thereby acquiring pronounced helical structural features. As the data further show, the protein binds to the membranes by an electrostatically driven mechanism, which requires a folded apo-state conformation and a negative membrane surface potential. Unfolded SOD1 molecules show no appreciable affinity to the membrane surfaces yielding a correlation between increased stability, i. e. occupancy of folded molecules and extend of membrane association. Since this trend opposes the correlation between decreased SOD1 stability and progression of neural damage, the results suggest that membrane association is not part of the ALS mechanism. An explanation could be that the observed membrane association of apo SOD1 is reversible and does not ‘bleed out’ in irreversible aggregation as observed for other precursors of protein-misfolding diseases.

  • 4.
    Alam, Md Khorshed
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vinklarek, Ivo
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sachl, Radek
    Fluorescence Studies of Lipid Distribution in Bilayers under Oxidative Stress2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal (Other academic)
  • 5. Areas, JAG
    et al.
    Glaubitz, C
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Watts, A
    A H-2 NMR study of the interaction of a type II myosin with charged bilayers2001In: BIOPHYSICAL JOURNAL, 2001, p. 536A-536AConference paper (Other academic)
  • 6. Beranová, Lenka
    et al.
    Humpolíčková, Jana
    Sýkora, Jan
    Benda, Aleš
    Cwiklik, Lukasz
    Jurkiewicz, Piotr
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hof, Martin
    Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations2012In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 14, no 42, p. 14516-14522Article in journal (Refereed)
    Abstract [en]

    Although there were experimental indications that phospholipid bilayers hydrated with D(2)O express different biophysical properties compared with hydration by ordinary H(2)O, a molecular concept for this behavior difference was only recently proposed by a molecular dynamics simulations study [T. Róg et al., J. Phys. Chem. B, 2009, 113, 2378-2387]. Here we attempt to verify those theoretical predictions by fluorescence measurements on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Specifically, we determine the water isotope effect on headgroup hydration and mobility, lateral lipid diffusion and lipid backbone packing. Time-dependent fluorescence shift experiments show significantly slower dynamics and lower hydration of the headgroup region for a bilayer hydrated with D(2)O, an observation in good agreement with the calculated predicted differences in duration of lipid-lipid and lipid-water bridges and extent of water penetration into the bilayer, respectively. The water isotope effect on the lipid order parameter of the bilayer core (measured by fluorescence anisotropy) and lateral diffusion of lipid molecules (determined by two-focus fluorescence correlation spectroscopy) is close to the experimental errors of the experiments, however also refers to slightly more rigid organization of phospholipid bilayers in heavy water. This study confirms the view that the water isotope effect can be particularly found in time-resolved physicochemical properties of the membrane. Together with the simulations our experiments provide a comprehensive, molecular view on the effect of D(2)O on phospholipid bilayers.

  • 7.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Misfolding of amyloidogenic proteins at membrane surfaces:  the impact of macromolecularcCrowding2007In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 48, p. 14848-14849Article in journal (Refereed)
    Abstract [en]

    The presence of inert macromolecular crowding agents mimics the situation in vivo where amyloidogenic proteins are released into an aqueous, congested intracellular environment. By using the amphiphatic Alzheimer A-protein as the model system, the presence of a three-dimensional macromolecular crowding environment enhanced significantly its misfolding behavior if charged membrane surfaces as two-dimensional aggregation templates were present.

  • 8.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrick
    Umeå University, Faculty of Science and Technology, Chemistry.
    Watts, Anthony
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Two Types of Alzheimer’s β-Amyloid (1–40) Peptide Membrane Interactions: Aggregation Preventing Transmembrane Anchoring Versus Accelerated Surface Fibril Formation2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 4, p. 1039-1049Article in journal (Refereed)
    Abstract [en]

    The 39–42 amino acid long, amphipathic amyloid-β peptide (Aβ) is one of the key components involved in Alzheimer's disease (AD). In the neuropathology of AD, Aβ presumably exerts its neurotoxic action via interactions with neuronal membranes. In our studies a combination of 31P MAS NMR (magic angle spinning nuclear magnetic resonance) and CD (circular dichroism) spectroscopy suggest fundamental differences in the functional organization of supramolecular Aβ1–40 membrane assemblies for two different scenarios with potential implication in AD: Aβ peptide can either be firmly anchored in a membrane upon proteolytic cleavage, thereby being prevented against release and aggregation, or it can have fundamentally adverse effects when bound to membrane surfaces by undergoing accelerated aggregation, causing neuronal apoptotic cell death. Acidic lipids can prevent release of membrane inserted Aβ1–40 by stabilizing its hydrophobic transmembrane C-terminal part (residue 29–40) in an α-helical conformation via an electrostatic anchor between its basic Lys28 residue and the negatively charged membrane interface. However, if Aβ1–40 is released as a soluble monomer, charged membranes act as two-dimensional aggregation-templates where an increasing amount of charged lipids (possible pathological degradation products) causes a dramatic accumulation of surface-associated Aβ1–40 peptide followed by accelerated aggregation into toxic structures. These results suggest that two different molecular mechanisms of peptide–membrane assemblies are involved in Aβ′s pathophysiology with the finely balanced type of Aβ–lipid interactions against release of Aβ from neuronal membranes being overcompensated by an Aβ–membrane assembly which causes toxic β-structured aggregates in AD. Therefore, pathological interactions of Aβ peptide with neuronal membranes might not only depend on the oligomerization state of the peptide, but also the type and nature of the supramolecular Aβ–membrane assemblies inherited from Aβ′s origin.

  • 9.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Borowik, T
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Interaction of Alzheimer's amyloid-beta peptide(1-40) with positively and negatively charged model membranes studied by circular dichroism and solid state NMR2005In: BIOPHYSICAL JOURNAL, ISSN 0006-3495, Vol. 88, no 1, p. 422A-Article in journal (Other academic)
    Abstract [en]

    Meeting Abstract

  • 10.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    CD and NMR studies of aggregation of amyloid-beta (1-40) peptide upon binding to model and raft membranes2003In: BIOPHYSICAL JOURNAL, 2003, p. 56A-56AConference paper (Other academic)
  • 11.
    Bokvist, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Mikhalyov, I.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Accelerated aggregation of amyloid-beta peptide on two-dimensional charged lipid membrane surfaces2004In: BIOPHYSICAL JOURNAL, ISSN 0006-3495, Vol. 86, no 1, p. 339A-339AArticle in journal (Other academic)
  • 12. Bonev, Boyan
    et al.
    Watts, Anthony
    Bokvist, Marcus
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Electrostatic peptide-lipid interactions of amyloid-β peptide and pentalysine with membrane surfaces monitored by 31P MAS NMR2001In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 3, no 14, p. 2904-2910Article in journal (Refereed)
    Abstract [en]

    High-resolution 31P magic angle spinning (MAS) NMR spectroscopy is presented as a direct and non-perturbing method for measuring changes in surface charge density occurring in mixed phospholipid membranes upon binding of charged surface-active peptides. 31P MAS NMR was used to investigate mixed lipid membranes of neutral phosphatidylcholine and negatively charged phosphatidylglycerol where the molar fraction of the charged lipid was varied from 0 to 1. The chemical shifts of the  individual membrane lipids showed a simple variation in response to changes in the fraction of the negatively charged component phosphatidylglycerol. Addition of the positively charged amyloid-β1-40 peptide, a key substance in Alzheimer's disease, resulted in changes in the isotropic chemical shifts of the membrane lipid phosphates in a way consistent with reduction in the negative surface charge of the mixed lipid bilayers. Binding of different amounts of the positively charged peptide pentalysine to L-α-dioleoylphosphatidylcholine/L-α-dioleoylphosphatidylglycerol(DOPC/DOPG) vesicles (2 : 1 molar ratio) also showed a systematic variation of both chemical shift values. These changes were described by a simple two-site model and indicate purely electrostatic binding of pentalysine.

  • 13.
    Bugaytsova, Jeanna A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björnham, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics. Swedish Defence Research Agency, 906 21 Umeå, Sweden.
    Chernov, Yevgen A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mahdavi, Jafar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. School of Life Sciences, CBS, University of Nottingham, NG7 2RD Nottingham, UK.
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ilver, Dag
    Moonens, Kristof
    Quintana-Hayashi, Macarena P.
    Moskalenko, Roman
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmidt, Alexej
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Åberg, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Koeniger, Verena
    Vikström, Susanne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ögren, Johan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine.
    Liu, Hui
    Goldman, Matthew D.
    Whitmire, Jeannette M.
    Åden, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Younson, Justine
    Kelly, Charles G.
    Gilman, Robert H.
    Chowdhury, Abhijit
    Mukhopadhyay, Asish K.
    Nair, G. Balakrish
    Papadakos, Konstantinos S.
    Martinez-Gonzalez, Beatriz
    Sgouras, Dionyssios N.
    Engstrand, Lars
    Unemo, Magnus
    Danielsson, Dan
    Suerbaum, Sebastian
    Oscarson, Stefan
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holgersson, Jan
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Landström, Maréne
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Eldridge, Angela M.
    Chromy, Brett A.
    Hansen, Lori M.
    Solnick, Jay V.
    Linden, Sara K.
    Haas, Rainer
    Dubois, Andre
    Merrell, D. Scott
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Remaut, Han
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E.
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence2017In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, no 3, p. 376-389Article in journal (Refereed)
    Abstract [en]

    The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

  • 14.
    Bugaytsova, Jeanna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björnhamn, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mahdavi, Jafar
    Ögren, Johan
    Ilver, Dag
    Gilman, Robert H
    Chowdhury, Abhijit
    The Swedish Institute for Control, Solna, Swede.
    Mukhopadhyay, Asish K
    Engstrand, Lars
    Oscarson, Stefan
    Kelly, Charles G
    Younson, Justine S
    Odenbreit, Stefan
    Solnick, Jay
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haas, Rainer
    Dubois, Andre
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Berg, Douglas E
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    pH regulated H. pylori adherence: implications for persistent infection and diseaseManuscript (preprint) (Other academic)
    Abstract [en]

    Helicobacter pylori’s BabA adhesin binds strongly to gastric mucosal ABH/Leb glycans on the stomach epithelium and overlying mucus, materials continuously shed into the acidic gastric lumen. Here we report that this binding is acid labile, acid inactivation is fully reversible; and acid lability profiles vary with BabA sequence and correlate with disease patterns. Isogenic H. pylori strains from the gastric antrum and more acidic corpus were identified that differed in acid lability of receptor binding and in sequence near BabA’s carbohydrate binding domain. We propose that reversible acid inactivation of receptor binding helps H. pylori avoid clearance by mucosal shedding, and that strain differences in acid lability affect tissue tropism and the spectrum of associated gastric diseases.

  • 15.
    Bugaytsova, Jeanna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chernov, Yevgen A
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mahdavi, Jafar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. School of Life Sciences, CBS, University of Nottingham, Nottingham, UK..
    Quintana-Hayashi, Macarena
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden..
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moskalenko, Roman
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Pathology, Medical Institute, State University, Sumy, Ukraine.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Université de Strasbourg, Institut de Chimie, Strasbourg, France.
    Moonens, Kristof
    Structural and Molecular Microbiology, VIB Department of Structural Biology, Belgium.
    Björnham, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics. FOI Totalförsvarets Forskningsinstitut, Umeå, Sweden..
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Königer, Verena
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU, Munich, Germany.
    Vikström, Susanne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmidt, Alexej
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ögren, Johan
    Umeå University, Faculty of Medicine, Department of Odontology.
    Ilver, Dag
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Liu, Hui
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Goldman, Matthew
    Department of Pediatrics, USUHS, Bethesda, MD, USA.
    Whitmire, Jeannette M
    Department of Microbiology and Immunology, USUHS, Bethesda, MD USA.
    Kelly, Charles G
    King's College London, Dental Institute, London, UK.
    Gilman, Robert H
    Department of International Health, John Hopkins School of Public Health, Baltimore, MD, USA.
    Chowdhury, Abhijit
    Centre for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education & Research, Kolkata, India.
    Mukhopadhyay, Asish K
    Division of Bacteriology, National Institute of Cholera and Enteric Diseases, Kolkata, India.
    Nair, Balakrish G
    Translational Health Science and Technology Institute, Haryana, India.
    Papadakos, Konstantinos S
    Hellenic Pasteur Institute, Athens, Greece.
    Martinez-Gonzalez, Beatriz
    Hellenic Pasteur Institute, Athens, Greece.
    Sgouras, Dionyssios N
    Hellenic Pasteur Institute, Athens, Greece.
    Engstrand, Lars
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Unemo, Magnus
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Danielsson, Dan
    Department of Laboratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden.
    Sebastian, Suerbaum
    Institute for Medical Microbiology and Hospital Epidemiology Hannover Medical School, Hannover, Germany.
    Oscarson, Stefan
    Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Dublin, Ireland.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holgersson, Jan
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Eldridge, Angela
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Chromy, Brett A
    Department of Pathology and Laboratory Medicine, University of California Davis School of Medicine, Sacramento, CA, USA.
    Hansen, Lori
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Solnick, Jay
    Departments of Medical Microbiology and Immunology, Center for Comparative Medicine, University of California Davis, Davis, CA, USA.
    Haas, Rainer
    Max von Pettenkofer Institute of Hygiene and Medical Microbiology, LMU Munich, Munich, Germany.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Lindén, Sara K
    Department of Biochemistry and Cell biology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Dubois, Andre
    Department of Medicine, USUHS, Bethesda, MD, USA.
    Merrell, D. Scott
    Department of Microbiology and Immunology, USUHS, Bethesda, MD, USA.
    Remaut, Han
    Structural and Molecular Microbiology, VIB Department of Structural Biology, VIB, Brussels, Belgium.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E
    Department of Medicine, University of California San Diego, La Jolla, CA, USA.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Acid Responsive Helicobacter pylori Adherence: Implications for Chronic Infection and DiseaseManuscript (preprint) (Other academic)
  • 16.
    Byström, Roberth
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Borowik, Tomasz
    Bokvist, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindström, Fredrick
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sani, Marc-Antoine
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Disordered proteins: Biological membranes as two-dimensional aggregation matrices2008In: Cell Biochemistry and Biophysics, ISSN 1085-9195, E-ISSN 1559-0283, Vol. 52, no 3, p. 175-189Article, review/survey (Refereed)
    Abstract [en]

    Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-β protein (Aβ), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane–protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.

  • 17.
    Byström, Roberth
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Chemistry.
    Oliveberg, Mikael
    Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Electrostatic interactions between negatively charged phospolipid membranes and SOD1 protein: Effect of charge changing fALS mutationsManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    The neurodegenerative disease amyotrophic lateral sclerosis (ALS) is closely connected to single site mutations of the Cu/Zn superoxide dismutase (SOD1) protein, whose pathological conversion into misfolded aggregates is a hallmark of ALS. To explore the impact of protein net charge changing ALS relevant SOD1 mutations on their ability to interact with neuronal membranes and the consequences for their folding behaviour, we studied by circular dichroism the conformational changes of the SOD1pWT, SOD1N86D and SOD1N86K species in their apo-state in the presence of increasing amounts of negatively charged lipid bilayers.. The results clearly indicate an electrostatically driven association process, where the association event induces a pronounced increase in the helical character of the pWT and the N86D species, characterized by long patient survival times. To the opposite, the charge reducing N86K mutation shows more pronounced β-like features in the presence of membranes in comparison to the other two species; an observation which most likely reflects its reduced stability in its apo-state in combination with a very fast ALS progression.

  • 18.
    Byström, Roberth
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersen, Peter M
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oliveberg, Mikael
    Stockholms universitet.
    SOD1 mutations targeting surface hydrogen bonds promote ALS without reducing apo-state stability2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 25, p. 19544-52Article in journal (Refereed)
    Abstract [en]

    In good accord with the protein-aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apo-SOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apo SOD1 is the elimination or introduction of a single charge, i.e., D76V/Y, D101N and N139D/K. The thermodynamic stability and folding behaviour of these mutants are indistinguishable from the wildtype control. Moreover, D101N is an outlier in the plot of stability loss vs. patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

  • 19.
    Byström, Roberth
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Andersen, Peter Munch
    Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Neurology.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Oliveberg, Mikael
    Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
    Identification of property outliers among ALS-associated SOD1 mutations: Common effect on surface hydrogen bondsManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    In good accord with the protein-aggregation hypothesis for neurodegenerative diseaseALS-associated SOD1 mutations are found to reduce structural stability or netrepulsive charge. Moreover there are weak indications that the ALS diseaseprogression is correlated with the degree of mutational impact on the SOD1 structure.A bottleneck for obtaining more conclusive information about these structure-diseaserelationships, however, is the large intrinsic variability in patient survival times andinsufficient disease statistics for the majority of ALS-provoking mutations. As analternative test of the structure-disease relationship we focus here on the SOD1 amutation that appears to be outliers in the data set. The results identify several ALSprovokingmutations whose only effect on apo SOD1 is the elimination orintroduction of a single charge, i.e., D76V/Y, D101N and N139D/K. Thethermodynamic stability and folding behaviour of these mutants are indistinguishablefrom the wildtype control, showing that structurally benign replacements of individualsurface charges are sufficient to trigger ALS. Moreover, D101N is a clear outlier inthe plot of stability loss vs. patient survival time by having too rapid diseaseprogression. Common to the identified mutations is that they truncate conserved saltlinksand/or H-bond networks in the functional loops IV or VII. The results show thatthe local impact of ALS-associated mutations on the SOD1 molecule can sometimesoverrun their global effects on stability and net repulsive charge, and point at theanalysis of property outliers as an efficient strategy for mapping out new ALSprovokingfeatures.

  • 20.
    Byström, Tomas
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lindblom, Göran
    Umeå University, Faculty of Science and Technology, Chemistry.
    Orientation of a polyleucine-based peptide in phosphatidylcholine bilayers of different thickness. Solid-state NMR and CD spectroscopy2003In: Colloids and Surfaces A: Physicochemical and Engineering Aspects, Vol. 228, no 1-3, p. 37-42Article in journal (Refereed)
    Abstract [en]

    A study was performed of the orientation and secondary structure of the peptide pLeuD11 (KKGL7DL[15N]WL9KKA) in phosphatidylcholine (PC) bilayers. The lipid bilayer thickness was varied by using PCs with monounsaturated acyl chains of different lengths, ranging from 14 to 24 carbon atoms. The peptide/lipid molar ratio was kept at 1:30 for all systems with water content of 50 wt.%. The secondary structure was determined by circular dichroism (CD) spectroscopy. The peptide adopted a transmembrane orientation in all bilayers, independent on their thickness. The location of the peptide was determined by 15N solid-state magic angle spinning (MAS) NMR spectroscopy, exploiting the effects of paramagnetic lanthanide ions at the membrane surface. From static solid-state 31P NMR spectroscopy measurements it was concluded that all lipid/peptide systems formed a lamellar liquid crystalline phase. As found by CD the distribution of secondary structures in the peptide changed only slightly for the different lipid membranes. The fraction of -helix was highest (≈60%) in bilayers with lipids, having an acyl chain length of 18 and 22 carbon atoms, while for lipids with 14 and 24 carbon atoms the helical content decreased slightly to about 50%. This reduction was accompanied by an increase in the fraction of β-like structures.

  • 21.
    Cardenas, Juan F
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lyophilized histidine investigated using X-ray photoelectron spectroscopy and cryogenics: Deprotonation in vacuum2005In: JOURNAL OF ELECTRON SPECTROSCOPY AND RELATED PHENOMENA, Vol. 148, no 2, p. 96-100Article in journal (Refereed)
    Abstract [en]

    Lyophilized histidine samples were investigated using X-ray photoelectron spectroscopy (XPS). Lyophilized samples were prepared from aqueous solutions at a pH in the range between similar to 1.5 and similar to 10, and with no further addition of electrolyte. The use of cryogenics allowed the determination of protonated to unprotonated molar ratios of sites in L-histidine, which correlates well with the dissociation constants of the residual amino acid sites. When cryogenics was not used deprotonation of the lyophilized samples occurred, where the degree and the total concentration of deprotonated sites correlates well with the formation constants and the decrease in Cl concentration, respectively. This later relation clearly indicates a correlation between deprotonation and the desorption of HCl from lyophilized samples. (c) 2005 Elsevier B.V. All rights reserved.

  • 22.
    Cardenas, Juan F
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    The influence of temperature and X-ray dose on the deprotonation of lyophilized phenylalanine during X-ray photoelectron spectroscopy2006In: Journal of Electron Spectroscopy and Related Phenomena, Vol. 152, p. 87-90Article in journal (Refereed)
    Abstract [en]

    Lyophilized phenylalanine (LP) samples were prepared from aqueous solutions at pH not, vert, similar 1.3 and subsequently analysed using X-ray photoelectron spectroscopy (XPS) in combination with cryogenics. When samples are measured at temperatures above not, vert, similar0 °C deprotonation occurs, which gradually proceeds with X-ray bombardment. In addition, deprotonation scales linearly with the difference between the Cl and the Na concentration, which strongly suggests that HCl sublimates from the sample.

  • 23.
    Cheregi, Otilia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Miranda, Hélder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers2015In: Journal of Photochemistry and Photobiology. B: Biology, ISSN 1011-1344, E-ISSN 1873-2682, p. 383-394Article in journal (Refereed)
    Abstract [en]

    The serine type Deg/HtrA proteases are distributed in a wide range of organisms from Escherichia coli to humans. The cyanobacterium Synechocystis sp. PCC 6803 possesses three Deg protease orthologues: HtrA, HhoA and HhoB. Previously we compared Synechocystis 6803 wild type cells exposed to mild or severe stress conditions with a mutant lacking all three Deg proteases and demonstrated that stress had strong impact on the proteomes and metabolomes [1]. To identify the biochemical processes, which this protease family is involved in, here we compared Synechocystis sp. PCC 6803 wild type cells with a mutant lacking all three Deg proteases grown under normal growth conditions (30 °C and 40 μmol photons m−2 s−1). Deletion of the Deg proteases lead to the down-regulation of proteins related to the biosynthesis of outer cell layers (e.g. the GDP mannose 4,6-dehydratase) and affected protein secretion. During the late growth phase of the culture Deg proteases were found to be secreted to the extracellular medium of the Synechocystis sp. PCC 6803 wild type strain. While cyanobacterial Deg proteases seem to act mainly in the periplasmic space, deletion of the three proteases influences the proteome and metabolome of the whole cell. Impairments in the outer cell layers of the triple mutant might explain the higher sensitivity toward light and oxidative stress, which was observed earlier by Barker and coworkers [2].

  • 24. Devanathan, Savitha
    et al.
    Salamon, Zdzislaw
    Lindblom, Göran
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Tollin, Gordon
    Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers2006In: FEBS Journal, ISSN 1742-464X, Vol. 273, no 7, p. 1389-402Article in journal (Refereed)
    Abstract [en]

    We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.

  • 25.
    Dingeldein, Artur P G
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorna, Sarka
    Hof, Martin
    Pedersen, Anders
    Karlsson, Göran
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    BCL-2 Family Proteins Effect on Mitochondrial-Mimicking Membrane Structure by Solid State NMR2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 251A-252AArticle in journal (Other academic)
    Abstract [en]

    Mitochondria are not only the cells' powerhouse, but also involved in their suicide via apoptosis. Key regulators of this pathway are members of the Bcl-2 protein family which interact with the outer mitochondrial membrane to modulate permeability and enable the release of apoptotic stimuli like cytochrome c. For a long time the mitochondrial membrane forming lipids have been seen as merely structural building units with proteins doing the actual work. This view changed in recent years, since lipids were shown to be also directly involved in apoptotic events e.g. under intracellular oxidative stress. Oxidized phospholipids (OxPls) generated under these stress conditions might trigger mitochondria-mediated apoptosis. Their presence in mitochondrial membranes can severely alter the properties of these membranes with yet unknown consequences regarding the formation of pores through membrane-mediated interplay with apoptotic Bax protein. We therefore devised a model system that embodies oxidative stress conditions by incorporating OxPls into mitochondria mimicking model membranes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) to study the impact of OxPls on apoptotic Bax-membrane interactions. To obtain molecular insight into hydrophobic fatty acid regions of membranes and their hydrophilic interface which is responsible for first protein-membrane contacts, we used differential scanning calorimetry (DSC) and solid state NMR spectroscopy. Upon incorporating OxPls with carboxyl (PoxnoPC) or aldehyde (PazePC) groups at their truncated sn-2-chains into our mitochondria model membranes, calorimetric and NMR measurements showed dramatic changes. 31P NMR experiments revealed major perturbation effects in these membranes; an effect which presumably elevates the membrane binding of apoptotic Bax to the charged membranes and its partial penetration, being a prerequisite for its final formation of pores which enable cytochrome c release from the mitochondrial interior. Currently structural studies of various Bax-lipid assemblies are ongoing.

  • 26.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 59A-60AArticle in journal (Other academic)
  • 27.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zhong, Xueyin
    Stoll, Raphael
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bax to the future – A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies2019In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, p. 20-26Article in journal (Refereed)
    Abstract [en]

    Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

  • 28.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oxidatively stressed mitochondria-mimicking membranes: a molecular insight into their organization during apoptosis2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 12, p. 2644-2654Article in journal (Refereed)
    Abstract [en]

    Mitochondria are crucially involved in the removal of eukaryotic cells by the intrinsic pathway of programmed cell death (apoptosis). The mitochondrion's outer membrane (MOM) is the platform where this pathway takes place. Upon oxidative stress triggering apoptotic action, the MOM undergoes permeabilization and release of cytochrome c, ultimately causing cell death. This membrane perforation is regulated not only by opposing members of the Bcl-2 protein family meeting at the MOM but also actively the membrane itself. Upon oxidative damage, the membrane undergoes severe reorganization causing an increase in cell death-causing apoptotic Bcl-2 proteins. To understand the active role of MOM, we provided a detailed molecular view of its structural and dynamic reorganization upon oxidative stress by solid-state C-13 MAS NMR (magic angle spinning nuclear magnetic resonance) accompanied by calorimetric studies. By focusing on MOM-like vesicles doped with oxidized lipid species, direct polarization C-13 MAS NMR provided a quantitative overview and identification of all lipid moieties across the membrane. H-1-C-1(3) cross polarization and insensitive nuclei enhanced by polarization transfer MAS NMR generated a dynamic - mobile versus restricted - membrane profile. Oxidized phospholipids significantly perturb the structural membrane organization and increase membrane dynamics. These perturbations are not uniformly distributed as the hydrophobic core is reflecting the melting of lipid chains and increase in molecular disorder directly, whereas the interface and headgroup region undergo complex dynamical changes, reflecting increased intra-molecular flexibility of these moieties. These changes are potentially crucial in augmenting pro-apoptotic action of proteins like Bax.

  • 29.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wacklin, Hanna P.
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal (Other academic)
  • 30.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorna, Sarka
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sachl, Radek
    Hof, Martin
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Apoptotic Bax at Oxidatively Stressed Mitochondrial Membranes: Lipid Dynamics and Permeabilization2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 10, p. 2147-2158Article in journal (Refereed)
    Abstract [en]

    Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using P-31 solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.

  • 31.
    Eriksson, Sylvia K
    et al.
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden.
    Kutzer, Michael
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Procek, Jan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Harryson, Pia
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden.
    Tunable membrane binding of the intrinsically disordered dehydrin Lti30, a cold-induced plant stress protein2011In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 23, no 6, p. 2391-2404Article in journal (Refereed)
    Abstract [en]

    Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30's proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest.

  • 32. Filippov, A. V.
    et al.
    Suleimanova, A. V.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Antsutkin, O. N.
    Effect of freezing on amyloid peptide aggregation and self-diffusion in an aqueous solution2008In: Colloid Journal, Vol. 70, no 4, p. 501-6Article in journal (Refereed)
    Abstract [en]

    Pulsed-field gradient 1H NMR is employed to investigate the self-diffusion of amyloid Aβ-peptide in an aqueous buffer solution (pH 7.44) with a protein concentration of 50 μmol at 20°C. The self-diffusion coefficient of the peptide in a freshly prepared solution corresponds to its monomeric form. The storage of the solution at 24°C causes part of the peptide molecules to form amyloid aggregates as soon as over 48 h. However, the 1H NMR echo signal typical of aggregated molecules is not observed because of their dense packing in the aggregates and a large mass of the latter. A freezing-fusion of the solution after the aggregation does not cause changes in the self-diffusion coefficients of the peptide. After a peptide solution free of amyloid aggregates is subjected to a freezing-fusion cycle, part of the peptide molecules also remains in the monomeric form in the solution, while another part forms amyloid aggregates, with a portion of the aggregated peptide molecules retaining a high rotational mobility with virtually absolute absence of a translational mobility. The results obtained are interpreted in terms of the formation of “porous aggregates” of amyloid fibrils, with “pores” having sizes comparable with those of peptide molecules, though, being larger than water molecules. Peptide molecules, which do not form fibrils, are captured in the pores. Temperature regime is shown to be of importance for the aggregation of amyloid peptides. In particular, freezing, which is traditionally considered to be a method for the prevention from or temporary interruption of aggregation, may itself lead to the formation of amorphous amyloid aggregates, which remain preserved in solutions after their unfreezing.

  • 33. Filippov, Andrei V
    et al.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Antzutkin, Oleg N
    Aggregation of amyloid Aβ(1-40) peptide in perdeuterated 2,2,2-trifluoroethanol caused by ultrasound sonication2010In: Magnetic Resonance in Chemistry, ISSN 0749-1581, E-ISSN 1097-458X, Vol. 48, no 6, p. 427-34Article in journal (Refereed)
    Abstract [en]

    Ultrasound sonication of protein and peptide solutions is routinely used in biochemical, biophysical, pharmaceutical and medical sciences to facilitate and accelerate dissolution of macromolecules in both aqueous and organic solvents. However, the impact of ultrasound waves on folding/unfolding of treated proteins, in particular, on aggregation kinetics of amyloidogenic peptides and proteins is not understood. In this work, effects of ultrasound sonication on the misfolding and aggregation behavior of the Alzheimer's Aβ(1-40)-peptide is studied by pulsed-field gradient (PFG) spin-echo diffusion NMR and UV circular dichroism (CD) spectroscopy. Upon simple dissolution of Aβ(1-40) in perdeuterated trifluoroethanol, CF3-CD2-OD (TFE-d3), the peptide is present in the solution as a stable monomer adopting -helical secondary structural motifs. The self-diffusion coefficient of Aβ(1-40) monomers in TFE-d3 was measured as 1.35 × 10-10 m2 s-1, reflecting its monomeric character. However, upon ultrasonic sonication for less than 5 min, considerable populations of A molecules (ca 40%) form large aggregates as reflected in diffusion coefficients smaller than 4.0 × 10-13 m2 s-1. Sonication for longer times (up to 40 min in total) effectively reduces the fraction of these aggregates in 1H PFG NMR spectra to ca 25%. Additionally, absorption below 230 nm increased significantly upon sonication treatment, an observation, which also clearly confirms the ongoing aggregation process of Aβ(1-40) in TFE-d3. Surprisingly, upon ultrasound sonication only small changes in the peptide secondary structure were detected by CD: the peptide molecules mainly adopt -helical motifs in both monomers and aggregates formed upon sonication.

  • 34. Filippov, Andrey
    et al.
    Munavirov, Bulat
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rudakova, Maya
    Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine2012In: Magnetic Resonance Imaging, ISSN 0730-725X, E-ISSN 1873-5894, Vol. 30, no 3, p. 413-421Article in journal (Refereed)
    Abstract [en]

    Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as "rafts," through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM-SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.

  • 35.
    Filippov, Andrey
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Sulejmanova, A
    Antzutkin, O
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Diffusion and aggregation of Alzheimer's A beta(1-40) peptide in aqueous trifluoroethanol solutions as studied by pulsed field gradient NMR2005In: APPLIED MAGNETIC RESONANCE, ISSN 0937-9347, Vol. 29, no 3, p. 439-49Article in journal (Refereed)
    Abstract [en]

    Pulsed field gradient nuclear magnetic resonance technique was applied to measure the self-diffusion coefficient of A beta(1-40) peptide in trifluoroethanol (TFE) and mixed solvent TFE-water (D2O) buffer (pD 7.8) at 293 K. The data were analyzed on the basis of the Stokes model and the hard-sphere approach was used to estimate self-diffusion coefficients. It was found that the extent of the A beta(1-40) aggregation in TFE solutions depends on the concentration of the peptide and the sample preparation protocol. After soft mixing, i.e., without any additional mechanical pretreatment of the peptide, the peptide is present in the monomeric form in TFE solutions. However, the additional water-bath sonication of the sample during the dissolution of A beta(1-40) in TFE enforces oligomerization of the peptide with the size of aggregates ranging from tetra- to hexamers. An increase of D2O in the mixed TFE-D2O solvent of up to 75% leads to the aggregation of the larger part of the peptide. However, the components of self-diffusion coefficients related to low-mass A beta(1-40). oligomers (dimers and trimers) were not observed in the diffusion decay curves. The most probable explanation is that dimers and trimers are not the principal intermediate species in the aggregation of A beta(1-40) peptide.

  • 36.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lipids and Cellular Membranes in Amyloid Diseases: Edited by Raz Jelinek2011In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 12, no 17, p. 2699-2700Article, book review (Refereed)
  • 37.
    Gröbner, Gerhard
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Burnett, Ian J
    Glaubitz, Clemens
    Choi, Gregory
    Mason, A James
    Watts, Anthony
    Observations of light-induced structural changes of retinal within rhodopsin2000In: Nature, Vol. 405, p. 810-3Article in journal (Refereed)
    Abstract [en]

    Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin1, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 Å) electron microscopy studies2, 3 confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation4, 5, 6. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method7. Significant structural changes are observed in the retinal following activation by light to the photo-activated MI state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

  • 38.
    Gröbner, Gerhard
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Glaubitz, C
    Williamson, PTF
    Hadingham, T
    Watts, A
    Structural features of membrane-bound amyloid-beta peptide determined by solid state NMR2001In: BIOPHYSICAL JOURNAL, 2001, p. 368A-368AConference paper (Other academic)
  • 39.
    Gröbner, Gerhard
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Glaubitz, Clemens
    Williamson, Philip T F
    Hadingham, Timothy
    Watts, Anthony
    Structural insight into the interaction of amyloid-β peptide with biological membranes by solid state NMR2001In: Perspectives on Solid State NMR in Biology, Kluwer Academic Publishers, Dordrecht , 2001, p. 203-14Chapter in book (Other academic)
  • 40.
    Gröbner, Gerhard
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Pandalai, S G
    Light triggered activation of the G-protein coupled photoreceptor rhodopsin: A structural and functional description2000In: Recent research developments in bioenergetics, Vol. 1, p. 103-15Article in journal (Refereed)
    Abstract [en]

    Rhodopsin, a 39 kDa photoreceptor is responsible for converting an incident photon of visible light into an optic nerve impulse. The protein belongs to the family of G-protein coupled receptors, which transduce chemical or optical signals across a cellular membrane. The ability of rhodopsin to convert light energy into a neuronal response is the result of isomerization of its chromophore retinal during the early primary photochemical events leading to activation of the protein. This activation is initiated by the photoisomerization of 11-cis retinal which is covalently attached to Lys-296 as a protonated Schiff base and gets slowly released from the protein as all-trans retinal. In order to understand the mechanism of conversion of light energy via this receptor protein into a final neuronal impulse, a precise elucidation of the structural organization of the chromophore binding site in the photoreceptor and its interaction with the chromophore after visual excitation is essential. Recently, various solid state NMR studies using nonperturbing spin reporters have provided an atomic level description of the overall orientation and structure of the chromophore within the binding pocket for various stages of the photoactivation, well ahead of any crystallographic efforts.

  • 41.
    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.

  • 42. Ivanov, A. V.
    et al.
    Pakusina, A. P.
    Ivanov, M. A.
    Sharutin, V. V.
    Gerasimenko, A. V.
    Antzutkin, O. N.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Forsling, W.
    Synthesis and single-crystal X-ray diffraction and CP/MAS 13C and 15N NMR study of tetraphenylantimony N,N-dialkyldithiocarbamate complexes: A manifestation of conformational isomerism2005In: Doklady Physical Chemistry, ISSN 0012-5016 (Print) 1608-3121 (Online), Vol. 401, no 4-6, p. 44-8Article in journal (Refereed)
  • 43. Jenske, Ramona
    et al.
    Lindström, Fredrick
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Camurus AB, Solvegatan 41, Ideon Science Park Gamma 1, SE-223 70 Lund, Sweden.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vetter, Walter
    Impact of free hydroxylated and methyl-branched fatty acids on the organization of lipid membranes2008In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 154, no 1, p. 26-32Article in journal (Refereed)
    Abstract [en]

    Differential scanning calorimetry (DSC) has been applied to study the effect of free hydroxylated and methyl-branched fatty acids on the physico-chemical properties of lipid membranes. First, the impact of free hydroxy fatty acids (HFAs) on dimyristoylphosphatidylcholine (DMPC) model membranes was monitored only as a function of chain length and position of the attached hydroxyl group. Second, racemic vs. enantiopure anteiso fatty acids (AFAs) and HFAs were investigated to address the question of which role does a fatty acid's chirality play on its membrane pertubing effect.

    The DSC thermograms revealed that the main gel to liquid–crystalline phase transition of the DMPC bilayers which results in a disordering effect of the lipid hydrocarbon chains was affected in different ways depending on the nature of the incorporated fatty acid. Long-chain 2- and 3-HFAs stabilized the gel phase by reducing the phase transition temperature (Tm), whereas short-chain HFAs and long-chain HFAs with the hydroxy group remote from the head group stabilized the more disordered liquid–crystalline state. Additionally, we observed that enantiopure (S)-14-methylhexadecanoic acid ((S)-a17:0) and (R)-2-hydroxy octadecanoic acid and the corresponding racemates had contrary effects upon incorporation into DMPC bilayers. In both cases, the pure enantiomers alleviated the liquid–crystalline state of the biological model membrane.

  • 44. 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.

  • 45. Khemtémourian, Lucie
    et al.
    Sani, Marc-Antoine
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bathany, Katell
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Dufourc, Erick J
    Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH42006In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 12, p. 58-64Article in journal (Refereed)
    Abstract [en]

    Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant -sheeted structures in water that convert progressively into -helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

  • 46. Koukalova, A.
    et al.
    Pokorna, S.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur P. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hof, M.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sachl, R.
    Oxidative stress as a modulator of BAX apoptotic activity2019In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 48, p. S170-S170Article in journal (Other academic)
    Abstract [en]

    Apoptosis is a regulated process of cell death that plays an important role in embryonic development, regulation of tissue homeostasis or removal of harmful cells in multicellular organisms. A failure in apoptosis can lead to severe pathological disorders including cancer, autoimmunity or neurodegenerative diseases. Mitochondria play a key role in the intrinsic apoptotic pathway, which is tightly regulated by Bcl-2 protein family. In response to cytotoxic stress, these proteins initiate cell death by permeabilization of the mitochondrial outer membrane (MOM) followed by the release of apoptotic factors, such as cytochrome c, which represents a point of no return in apoptotic progression. BAX protein, a member of the pro-apoptotic Bcl-2 family with a pore forming activity, is a critical effector of the mitochondrial cell death pathway. Upon interaction with BH3-only proteins, cytosolic BAX undergoes conformational activation and translocation resulting in MOM permeabilization. However, the underlying mechanism controlling this membrane-associated BAX action is still poorly understood. Recent findings suggest that the activity of BAX protein is significantly regulated by the lipid composition of the MOM. In particular, oxidized phospholipids (OxPL) that are generated under oxidative stress conditions seem to directly influence BAX membrane penetration and its activity [1,2].

    By employing single molecule fluorescence spectroscopy techniques and various dye leakage assays we study the BAX membrane interactions and its activity in in vitro system that makes use of synthetic giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) doped with varying amounts of OxPL species, which mimics oxidative stress conditions. Dye leakage assays revealed that BAX activity is sensitive to the type and concentration of different OxPL species in the MOM-mimicking vesicles. Higher levels of oxidized phospholipids in MOM mimicking model lipid bilayer significantly enhance membrane affinity and partial penetration of full length BAX. In addition, solid state NMR studies and calorimetric experiments on the lipid vesicles revealed that the presence of OxPL disrupts the membrane organization enabling BAX to penetrate into the membrane.

  • 47. Koukalova, Alena
    et al.
    Amaro, Mariana
    Aydogan, Gokcan
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Williamson, Philip T. F.
    Mikhalyov, Ilya
    Hof, Martin
    Sachl, Radek
    Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5460Article in journal (Refereed)
    Abstract [en]

    It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)-and cholesterol (Chol)-driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Forster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solidstate wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

  • 48. Kristiansen, PE
    et al.
    Fimland, G
    Nissen-Meyer, J
    Borowik, T
    Lindstrom, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bokvist, Marcus
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Chemistry.
    Mode of action of antimicrobial sakacin P peptides with biological membranes studied by calorimetry, circular dichroism and biological MAS NMR2004In: BIOPHYSICAL JOURNAL, ISSN 0006-3495, Vol. 86, no 1, p. 339A-339AArticle in journal (Other academic)
  • 49.
    Lidman, Martin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorná, Šárka
    Šachl, Radek
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hof, Martin
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The Role of Lipids in Regulation of Programmed Cell Death2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 473A-473AArticle in journal (Refereed)
  • 50.
    Lidman, Martin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorná, Šárka
    Dingeldein, Artur P. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Šachl, Radek
    Hof, Martin
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 6, p. 1288-1297Article in journal (Refereed)
    Abstract [en]

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly 15N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.

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