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  • 1.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Delivering catalase to yeast peroxisomes using cell-penetrating peptidesIn: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658Article in journal (Refereed)
  • 2.
    Bartish, Galyna
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Moradi, Hossein
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Nygård, Odd
    Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 20, p. 5285-5297Article in journal (Refereed)
    Abstract [en]

    Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

  • 3.
    Belotserkovsky, Jaroslav M.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dabbs, Eric R.
    Isaksson, Leif A.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 18, p. 3508-3517Article in journal (Refereed)
    Abstract [en]

    A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h) 18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.

  • 4.
    Belotserkovsky, Jaroslav M.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isak, Georgina I.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isaksson, Leif A.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 10, p. 1745-1756Article in journal (Refereed)
    Abstract [en]

    Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

  • 5.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kurnik, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 3, p. 814-824Article in journal (Refereed)
    Abstract [en]

    The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

  • 6. Boukhelifa, Malika
    et al.
    Moza, Monica
    Johansson, Thomas
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Rachlin, Andrew
    Parast, Mana
    Huttelmaier, Stefan
    Roy, Partha
    Jockusch, Brigitte, M
    Carpen, Olli
    Karlsson, Roger
    Otey, Carol, A
    The proline-rich protein palladin is a binding partner for profilin2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 1, p. 26-33Article in journal (Refereed)
    Abstract [en]

    Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), α-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

  • 7. Brown, Christian
    et al.
    Szpryngiel, Scarlett
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuang, Guanglin
    Srivastava, Vaibhav
    Ye, Weihua
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    McKee, Lauren S.
    Yaoquan, Tu
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bulone, Vincent
    Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 16, p. 3072-3088Article in journal (Refereed)
    Abstract [en]

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

  • 8.
    Dahlroth, Sue-Li
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gurmu, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haas, Juergen
    Erlandsen, Heidi
    Nordlund, Pär
    Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 22, p. 6636-6645Article in journal (Refereed)
    Abstract [en]

    The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein–Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 Å resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein–protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.

  • 9.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pierattelli, Roberta
    Banci, Lucia
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High-resolution NMR studies of the sinc-binding site of the Alzheimer’s Aβ-peptide2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 1, p. 46-59Article in journal (Refereed)
  • 10. Egeblad, Louise
    et al.
    Welin, Martin
    Johansson, Andreas
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, Liya
    Flodin, Susanne
    Nyman, Tomas
    Trésaugues, Lionel
    Kotenyova, Tetyana
    Johansson, Ida
    Eriksson, Staffan
    Eklund, Hans
    Nordlund, Pär
    Structural and functional studies of the human phosphoribosyltransferase domain containing protein 12010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 23, p. 4920-30Article in journal (Refereed)
    Abstract [en]

    Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.

  • 11.
    Ge, Changrong
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University Libre Brussels, Belgium; Karolinska Institutet, Sweden.
    Gómez Llobregat, Jordi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Skwark, Marcin J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Aalto University, Finland.
    Ruysschaert, Jean-Marie
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindén, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane remodeling capacity of a vesicle-inducing glycosyltransferase2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 16, p. 3667-3684Article in journal (Refereed)
    Abstract [en]

    Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.

  • 12. Johansson, Renzo
    et al.
    Torrents, Eduard
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sprenger, Janina
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states: an unusual flavodoxin2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 20, p. 4265-4277Article in journal (Refereed)
    Abstract [en]

    The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4-10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

  • 13.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Chenge
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. chenge.li@dbb.su.se.
    Montigny, Cedric
    le Maire, Marc
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, p. 5398-5407Article in journal (Refereed)
    Abstract [en]

    Recombinant Ca2+-ATPase was expressed in Saccharomycescerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200g of similar to 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca(2)E1P and Ca(2)E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca(2)E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

  • 14.
    Lind, Jesper
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Emma
    Perálvarez-Marín, Alex
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holmlund, Anna
    Jörnvall, Hans
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural features of proinsulin C-peptide oligomeric and amyloid states2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 18, p. 3759-68Article in journal (Refereed)
    Abstract [en]

    The formation and structure of proinsulin C-peptide oligomers has been investigated by PAGE, NMR spectroscopy and dynamic light scattering. The results obtained show that C-peptide forms oligomers of different sizes, and that their formation and size distribution is altered by salt and divalent metal ions, which indicates that the aggregation process is mediated by electrostatic interactions. It is further demonstrated that the size distribution of the C-peptide oligomers, in agreement with previous studies, is altered by insulin, which supports a physiologically relevant interaction between these two peptides. A small fraction of oligomers has previously been suggested to be in equilibrium with a dominant fraction of soluble monomers, and this pattern also is observed in the present study. The addition of modest amounts of sodium dodecyl sulphate at low pH increases the relative amount of oligomers, and this effect was used to investigate the details of both oligomer formation and structure by a combination of biophysical techniques. The structural properties of the SDS-induced oligomers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR spectroscopy, demonstrate that soluble aggregates are predominantly in β-sheet conformation, and that the oligomerization process shows characteristic features of amyloid formation. The formation of large, insoluble, β-sheet amyloid-like structures will alter the equilibrium between monomeric C-peptide and oligomers. This leads to the conclusion that the oligomerization of C-peptide may be relevant also at low concentrations.

  • 15.
    Nordlund, Stefan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    ADP-ribosylation, a mechanism regulating nitrogenase activity2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 15, p. 3484-3490Article, review/survey (Refereed)
    Abstract [en]

    Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase, is controlled by reversible mono-ADP-ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe-protein is ADP-ribosylated and de-ADP-ribosylated by dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.

  • 16.
    Pettersson, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ye, Weihua
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jakob, Mario
    Tannert, Franzisca
    Klösgen, Ralf Bernd
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 10, p. 1886-1906Article in journal (Refereed)
    Abstract [en]

    The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated core TatA' from Arabidopsisthaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using N-15 relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

  • 17.
    Pisareva, Tatiana
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shumskaya, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maddalo, Gianluca
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Ilag, Leopold
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Norling, Birgitta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteomics of Synechocystis sp. PCC 6803 Identification of novel integral plasma membrane proteins: Identification of novel integral plasma membrane proteins2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 3, p. 791-804Article in journal (Refereed)
    Abstract [en]

    The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI-TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.

  • 18.
    Runesson, Johan
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Saar, Indrek
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Järv, Jaak
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Novel peptide agonists, favoring galanin receptor type 2 over galanin receptor type 1 and 32009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, p. 164-164Article in journal (Refereed)
    Abstract [en]

    The galanin peptide family and its three receptors have with compelling evidence been implicated in a variety of human disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family although, so far, no therapeutics have emerged past the animal model stage. In the current study we present data on receptor binding and functional response from novel galanin receptor type 2 (GalR2) selective chimeric peptides, including the M1145 peptide which show more than 90-fold higher affinity for galanin receptor type 2 over galanin receptor type 1 and a 76-fold higher affinity over galanin receptor type 3. Furthermore, the peptide produces an agonistic effect in vitro seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family. Preliminary data on peptides further exploring the usage of N-terminal extension shows even higher preferentiality towards the GalR2 and opens new possibilities to clarify the galanin system as a putative drug target.

  • 19.
    Sheng, Xiang
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Lind, Maria E. S.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Himo, Fahmi
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Theoretical study of the reaction mechanism of phenolic acid decarboxylase2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, no 24, p. 4703-4713Article in journal (Refereed)
    Abstract [en]

    The cofactor-free phenolic acid decarboxylases (PADs) catalyze the non-oxidative decarboxylation of phenolic acids to their corresponding p-vinyl derivatives. Phenolic acids are toxic to some organisms, and a number of them have evolved the ability to transform these compounds, including PAD-catalyzed reactions. Since the vinyl derivative products can be used as polymer precursors and are also of interest in the food-processing industry, PADs might have potential applications as biocatalysts. We have investigated the detailed reaction mechanism of PAD from Bacillus subtilis using quantum chemical methodology. A number of different mechanistic scenarios have been considered and evaluated on the basis of their energy profiles. The calculations support a mechanism in which a quinone methide intermediate is formed by protonation of the substrate double bond, followed by C-C bond cleavage. A different substrate orientation in the active site is suggested compared to the literature proposal. This suggestion is analogous to other enzymes with p-hydroxylated aromatic compounds as substrates, such as hydroxycinnamoyl-CoA hydratase-lyase and vanillyl alcohol oxidase. Furthermore, on the basis of the calculations, a different active site residue compared to previous proposals is suggested to act as the general acid in the reaction. The mechanism put forward here is consistent with the available mutagenesis experiments and the calculated energy barrier is in agreement with measured rate constants. The detailed mechanistic understanding developed here might be extended to other members of the family of PAD-type enzymes. It could also be useful to rationalize the recently developed alternative promiscuous reactivities of these enzymes.

  • 20.
    Surkov, Serhiy
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Nilsson, Hanna
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Rasmussen, Louise CV
    Department of Molecular Biology, Aarhus university.
    Sperling-Petersen, Hans U
    Department of Molecular Biology, Aarhus university.
    Isaksson, Leif
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 11, p. 2428-2439Article in journal (Refereed)
    Abstract [en]

    Translation initiation factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation initiation on the ribosome remains unclear, even though the factor has several known functions, one of them being RNA chaperone activity. In this study, we analyzed translational gene expression mutant variants of IF1 with amino acid substitutions, R40D and R69L, using two different reporter gene systems. The strains with them mutant IF1 gave higher reporter gene expression than the control strain. The extent of this effect was dependent on the composition of the translation initiation region. The Shine–Dalgarno (SD) sequence, AU-rich elements upstream of the SD sequence and the region between the SD sequence and the initiation codon are important for the magnitude of this effect. The data suggest that the wild-type form of IF1 has a translation initiation region-dependent inhibitory effect on translation initiation. Kasugamycin is an antibiotic that blocks translation initiation. Addition of kasugamycin to growing wild-type cells increases reporter gene expression in a very similar way to the altered IF1, suggesting that the and kasugamycin affect some related step in translation initiation. Genetic knockout of three proteins (YggJ, BipA, and CspA) that are known to interact with RNA causes partial suppression of the IF1-dependent cold sensitivity.

  • 21.
    Wahlström, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hugonin, Loïc
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perálvarez-Marín, Alex
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Jüri
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Secondary structure conversions of Alzheimer’s Aβ(1–40) peptide induced by membrane-mimicking detergents2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 20, p. 5117-5128Article in journal (Refereed)
    Abstract [en]

    The amyloid β peptide (Aβ) with 39–42 residues is the major component of amyloid plaques found in brains of Alzheimer’s disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Aβ aggregation involves a conformational change of the peptide structure to β-sheet. In the present study, we report on the effect of detergents on the structure transitions of Aβ, to mimic the effects that biomembranes may have. In vitro, monomeric Aβ(1–40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Aβ(1–40) is converted to β-sheet, as observed by CD at 3 °C and 20 °C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Aβ(1–40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal β-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with α-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.

  • 22.
    Ye, Weihua
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Spånning, Erika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Unnerståle, Sofia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gotthold, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 19, p. 3738-3748Article in journal (Refereed)
    Abstract [en]

    Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form a-helical structure in one region, S6F27, and a very weak beta-strand propensity for Q34Q38. The a-helical structured region has an amphiphilic character and a f??ff motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9V28, overlaps with the amphiphilic region, and the other comprises residues L30Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. Database The backbone chemical shift assignments for ThrRS-dTP(260) have been deposited with the Biological Magnetic Resonance Bank (BMRB) under the accession code 18248 Structured digital abstract ThrRS-dTP and Tom20-4 bind by nuclear magnetic resonance (View interaction)

  • 23. Zhai, Jianjun
    et al.
    Ström, Anna-Lena
    University of Kentucky, USA.
    Kilty, Renee
    Venkatakrishnan, Priya
    White, James
    Everson, William V.
    Smart, Eric J.
    Zhu, Haining
    Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.2009In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 12, p. 3308-3323Article in journal (Refereed)
    Abstract [en]

    Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.

1 - 23 of 23
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