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  • 1. Anko, Maja
    et al.
    Majhenc, Janja
    Kogej, Ksenija
    Sillard, Rannard
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anderluh, Gregor
    Zorko, Matjaz
    Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane2012In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 3, p. 915-924Article in journal (Refereed)
    Abstract [en]

    The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PR and PF6 observed in previous studies.

  • 2.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindholm, Ljubica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonny
    Edwards, Katarina
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes:  2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, p. 1862-1870Article in journal (Refereed)
    Abstract [en]

    The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

  • 3. Arukuusk, Piret
    et al.
    Paernaste, Ly
    Oskolkov, Nikita
    Copolovici, Dana-Maria
    Margus, Helerin
    Padari, Kaert
    Moell, Kaidi
    Maslovskaja, Julia
    Tegova, Radi
    Kivi, Gaily
    Tover, Andres
    Pooga, Margus
    Ustav, Mart
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 5, p. 1365-1373Article in journal (Refereed)
    Abstract [en]

    Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

  • 4.
    Björkholm, Patrik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ernst, Andreas M.
    Hacke, Moritz
    Wieland, Felix
    Bruegger, Britta
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Identification of novel sphingolipid-binding motifs in mammalian membrane proteins2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 8, p. 2066-2070Article in journal (Refereed)
    Abstract [en]

    Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.

  • 5.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Mathias
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Analysing DHPC/DMPC bicelles by diffusion NMR and multivariate decomposition2015In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, no 11, p. 2910-2917Article in journal (Refereed)
    Abstract [en]

    Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many uncertainties remain concerning the details of the phase behaviour of these mixtures and the morphology of the formed lipid assemblies. Here we used nuclear magnetic resonance (NMR) diffusion data in combination with the multivariate processing method speedy component resolution (SCORE) to analyse mixtures of 1,2-dihexanoyl-snglycero-3-phosphocholine (DHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the relative concentration q = [DMPC]/[DHPC] = 0.5 at total lipid concentrations ranging from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coefficients for lipid concentrations in the range 15 to 300 mM, although at high concentrations (250-300 mM), non-negativity constraints or overfactoring was required to successfully decompose the data. At 50-300 mM total lipid concentration, the radii estimated from the diffusion coefficient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q = 0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may reliably be assumed to adopt the 'classical' bicelle morphology. The study clearly demonstrates the usefulness of our approach for accurately determining physical properties of complex mixtures such as bicelles. Both reliable diffusion coefficients and chemical shifts can be derived from overlapping data. This should prove useful for analysing the behaviour of other, more complex, lipid mixtures.

  • 6. Bodor, Andrea
    et al.
    Kover, Katalin E.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane interactions in small fast-tumbling bicelles as studied by P-31 NMR2015In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, no 3, p. 760-766Article in journal (Refereed)
    Abstract [en]

    Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used P-31 NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and P-31 T-2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T-2 relaxation are observed at higher temperatures. A comparison of P-31 T-1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T-1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules - in particular for the negatively charged DMPG - while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide-membrane interactions.

  • 7.
    Botelho, Salome C.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger R.
    Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues2015In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, no 2, p. 615-621Article in journal (Refereed)
    Abstract [en]

    Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.

  • 8.
    Castro, Vasco
    et al.
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Stevensson, Baltzar
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Dvinskikh, Sergey V.
    Division of Physical Chemistry, Royal Institute of Technology, SE-10044 Stockholm, Sweden.
    Högberg, Carl-Johan
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Lyubartsev, Alexander P.
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Zimmermann, Herbert
    Department of Biophysics, Max-Planck-Institut für Medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany.
    Sandström, Dick
    Bruker BioSpin Scandinavia AB, Polygonvägen 79, SE-187 66 Täby, Sweden.
    Maliniak, Arnold
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    NMR investigations of interactions between anesthetics and lipid bilayers2008In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1178, no 11, p. 2604-2611Article in journal (Refereed)
    Abstract [en]

    Interactions between anesthetics (lidocaine and short chain alcohols) and lipid membranes formed by dimyristoylphosphatidylcholine (DMPC) were studied using NMR spectroscopy. The orientational order of lidocaine was investigated using deuterium NMR on a selectively labelled compound whereas segmental ordering in the lipids was probed by two-dimensional 1H–13C separated local field experiments under magic-angle spinning conditions. In addition, trajectories generated in molecular dynamics (MD) computer simulations were used for interpretation of the experimental results. Separate simulations were carried out with charged and uncharged lidocaine molecules. Reasonable agreement between experimental dipolar interactions and the calculated counterparts was observed. Our results clearly show that charged lidocaine affects significantly the lipid headgroup. In particular the ordering of the lipids is increased accompanied by drastic changes in the orientation of the P–N vector in the choline group.

  • 9.
    Eiríksdóttir, Emelía
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Konate, Karidia
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Divita, Gilles
    Deshayes, Sébastien
    Secondary Structure of Cell-Penetrating Peptides Controls Membrane Interaction and Insertion2010In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 6, p. 1119-1128Article in journal (Refereed)
    Abstract [en]

    The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.

  • 10.
    Guterstam, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, p. 2509-2517Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

  • 11.
    Howard, Rebecca J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Carnevale, Vincenzo
    Delemotte, Lucie
    Hellmich, Ute A.
    Rothberg, Brad S.
    Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 4, p. 927-942Article, review/survey (Refereed)
    Abstract [en]

    Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process for example with neuroactive drugs demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin Mcllwain.

  • 12.
    Jafferali, Mohammed Hakim
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vijayaraghavan, Balaje
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Crafoord, Ellinor
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gudise, Santhosh
    Stockholm University, Faculty of Science, Department of Neurochemistry. Karolinska Institutet, Sweden.
    Larsson, Veronica J.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 10, p. 2399-2403Article in journal (Refereed)
    Abstract [en]

    Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.

  • 13. Juks, Carmen
    et al.
    Padari, Kärt
    Margus, Helerin
    Kriiska, Asko
    Etverk, Indrek
    Arukuusk, Piret
    Koppel, Kaida
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Oxford, UK.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    The role of endocytosis in the uptake and intracellular trafficking of PepFect14-nucleic acid nanocomplexes via class A scavenger receptors2015In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, no 12, p. 3205-3216Article in journal (Refereed)
    Abstract [en]

    Cell penetrating peptides are efficient tools to deliver various bioactive cargos into cells, but their exactfunctioning mechanism is still debated. Recently, we showed that a delivery peptide PepFect14 condenses oligonudeotides (ON) into negatively charged nanocomplexes that are taken up by cells via class A scavenger receptors (SR-As). Here we unraveled the uptake mechanism and intracellular trafficking of PF14-ON nanocomplexes in HeLa cells. Macropinocytosis and caveolae-mediated endocytosis are responsible for the intracellular functionality of nucleic acids packed into nanocomplexes. However, only a negligible fraction of the complexes were trafficked to endoplasmic reticulum or Golgi apparatus the common destinations of caveolar endocytosis. Neither were the PF14-SCO nanocomplexes routed to endo-lysosomal pathway, and they stayed in vesicles with slightly acidic pH, which were not marked with LysoSensor. Naked ON, in contrary, was rapidly targeted to acidic vesicles and lysosomes. The transmission electron microscopy analysis of interactions between SR-As and PF14-ON nanocomplexes on ultrastructural level revealed that nanocomplexes localized on the plasma membrane in close proximity to SR-As and their colocalization is retained in cells, suggesting that PF14-ON complexes associate with targeted receptors.

  • 14. Leal, Cecilia
    et al.
    Sandstroem, Dick
    Stockholm University.
    Nevsten, Pernilla
    Topgaard, Daniel
    Local and translational dynamics in DNA-lipid assemblies monitored by solid-state and diffusion NMR2008In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1778, no 1, p. 214-228Article in journal (Refereed)
    Abstract [en]

    The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by P-31-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved C-13 spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the H-1 dipolar linewidth (WISE) or the H-1-C-13 dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA-lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing. 

  • 15.
    Liebau, Jobst
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fu, Biao
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brown, Christian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 3, p. 683-690Article in journal (Refereed)
    Abstract [en]

    Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.

  • 16.
    Liebau, Jobst
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pettersson, Pontus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zuber, Philipp
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ariöz, Candan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Chalmers University of Technology, Sweden.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fast-tumbling bicelles constructed from native Escherichia coli lipids2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 9, p. 2097-2105Article in journal (Refereed)
    Abstract [en]

    Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by P-31 and C-13 NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR.

  • 17.
    Lyubartsev, Alexander P.
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Rabinovich, Alexander L.
    Force Field Development for Lipid Membrane Simulations2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 10, p. 2483-2497Article in journal (Refereed)
    Abstract [en]

    With the rapid development of computer power and wide availability of modelling software computer simulations of realistic models of lipid membranes, including their interactions with various molecular species, polypeptides and membrane proteins have become feasible for many research groups. The crucial issue of the reliability of such simulations is the quality of the force field, and many efforts, especially in the latest several years, have been devoted to parametrization and optimization of the force fields for biomembrane modelling. In this review, we give account of the recent development in this area, covering different classes of force fields, principles of the force field parametrization, comparison of the force fields, and their experimental validation. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Rog.

  • 18.
    Madani, Fatemeh
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abdo, Rania
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hirose, Hisaaki
    Futaki, Shiroh
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 4, p. 1198-1204Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  • 19. Maeger, Imre
    et al.
    Langel, Kent
    Lehto, Taavi
    Eiriksdottir, Emelia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides2012In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 3, p. 502-511Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.

  • 20.
    Magzoub, Mazin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kilk, Kalle
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles2001In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1512, no 1, p. 77-89Article in journal (Refereed)
    Abstract [en]

    Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp (‘penetratin’), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% α-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% β-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of α-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.

  • 21. Metola, Ane
    et al.
    Bouchet, Ana M.
    Alonso-Marino, Marian
    Diercks, Tammo
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Goni, Felix M.
    Viguera, Ana R.
    Purification and characterization of the colicin A immunity protein in detergent micelles2017In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 11, p. 2181-2192Article in journal (Refereed)
    Abstract [en]

    The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.

  • 22.
    Mäger, Imre
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Kent
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay2010In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 3, p. 338-343Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.

  • 23. Pae, Janely
    et al.
    Liivamagi, Laura
    Lubenets, Dmitri
    Arukuusk, Piret
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 8, p. 1860-1867Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

  • 24.
    Sabanov, Victor
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Ca2+-independent effects of BAPTA and EGTA on single-channel Cl- currents in brown adipocytes2007In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1768, no 11, p. 2714-2725Article in journal (Refereed)
    Abstract [en]

    The Cl channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl channels (ORCC). To study tentative Ca2+ regulation of these channels, we attempted to control Ca2+ levels at the cytoplasmic side of the inside-out membrane patches with Ca2+-chelating agents. However, we found that the commonly used Ca2+-chelators EGTA and BAPTA by themselves influenced the Cl channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca2+-chelators (acting from the cytoplasmic side) on the single Cl channel currents in patch-clamp experiments. Using fixed (1–2 mM) concentrations of chelators, two types of Cl channels were identified, as discriminated by their reaction to the Ca2+-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca2+. The anionic classical Cl channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca2+ level control in themselves could fundamentally affect the behaviour of Cl channels.

  • 25. Shimoji, Miyuki
    et al.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Neve, Etienne
    Maksel, Danuta
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Imreh, Gabriela
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Morgenstern, Ralf
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 12017In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 2, p. 238-244Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.

  • 26.
    Smirnova, Irina A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Moscow State University, Russian Federation.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Fei
    Björck, Markus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Östbye, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University, United States.
    von Ballmoos, Christoph
    Lander, Gabriel C.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation of yeast complex IV in native lipid nanodiscs2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, p. 2984-2992Article in journal (Refereed)
    Abstract [en]

    We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

  • 27.
    ter Beek, Josy
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kahle, Maximilian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modulation of protein function in membrane mimetics: Characterization of P. denitrificans cNOR in nanodiscs or liposomes2017In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 10, p. 1951-1961Article in journal (Refereed)
    Abstract [en]

    For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitnficans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O-2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O-2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O-2, and found that the pK(a) of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

  • 28.
    Unnerståle, Sofia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural characterization of AS1-membrane interactions from a subset of HAMP domains2011In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1808, no 10, p. 2403-2412Article in journal (Refereed)
    Abstract [en]

    HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in alpha-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic alpha-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

  • 29.
    Vasconcelos, Luis
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Tõnis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Radoi, Vlad
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vukojević, Vladana
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 2, p. 491-504Article in journal (Refereed)
    Abstract [en]

    Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

  • 30.
    Vijayaraghavan, Balaje
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo A.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Bergqvist, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gupta, Amit J.
    Sousa, Paulo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin2018In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 6, p. 1326-1334Article in journal (Refereed)
    Abstract [en]

    Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope.

  • 31.
    Zhang, Xuan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oglecka, Kamila
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sandgren, Staffan
    Belting, Mattias
    Esbjorner, Elin K.
    Norden, Bengt
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dual functions of the human antimicrobial peptide LL-37-Target membrane perturbation and host cell cargo delivery2010In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, no 12, p. 2201-2208Article in journal (Refereed)
    Abstract [en]

    The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced alpha-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel alpha-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.

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