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  • 1.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed)
    Abstract [en]

    Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

  • 2.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Rafstedt, Therese
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Supuran, Claudiu T
    University of Florence.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 23, p. 5358-5364Article in journal (Refereed)
    Abstract [en]

    Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.

  • 3. Andersson, D.
    et al.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Cofactor-induced refolding: Refolding of molten globule carbonic anhydrase induced by Zn(II) and Co(II)2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 9, p. 2653-2661Article in journal (Refereed)
    Abstract [en]

    The stability versus unfolding to the molten globule intermediate of bovine carbonic anhydrase II (BCA II) in guanidine hydrochloride (GuHCl) was found to depend on the metal ion cofactor [Zn(II) or Co(II)], and the apoenzyme was observed to be least stable. Therefore, it was possible to find a denaturant concentration (1.2 M GuHCl) at which refolding from the molten globule to the native state could be initiated merely by adding the metal ion to the apo molten globule. Thus, refolding could be performed without changing the concentration of the denaturant. The molten globule intermediate of BCA II could still bind the metal cofactor. Cofactor-effected refolding from the molten globule to the native state can be summarized as follows: (1) initially, the metal ion binds to the molten globule, (2) compaction of the metal-binding site region is then induced by the metal ion binding, (3) a functioning active center is formed, and (4) finally, the native tertiary structure is generated in the outer parts of the protein.

  • 4.
    Belogurov, G A
    et al.
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Fabrichniy, I P
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Pohjanjoki, P
    Department of Biochemistry, University of Turku, Turku, Finland.
    Kasho, V N
    Center for Ulcer Research and Education, Department of Medicine, University of California, Los Angeles, California, USA.
    Lehtihuhta, E
    Department of Biochemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Cooperman, B S
    Department of Chemistry, University of Pennsylvania, Pennsylvania, USA.
    Goldman, A
    Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
    Baykov, A A
    A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
    Lahti, R
    Department of Biochemistry, University of Turku, Turku, Finland.
    Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 45, p. 13931-13938Article in journal (Refereed)
    Abstract [en]

    Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

  • 5.
    Belogurov, Georgiy A
    et al.
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Malinen, Anssi M
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Turkina, Maria V
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Jalonen, Ulla
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Rytkönen, Kalle
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Baykov, Alexander A
    A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow, Russia.
    Lahti, Reijo
    Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
    Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 6, p. 2088-2096Article in journal (Refereed)
    Abstract [en]

    Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.

  • 6. Chen, H
    et al.
    Nyström, Fredrik
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology. Östergötlands Läns Landsting, MKC-2, GE: endomed.
    Dong, LQ
    Cong, L
    Li, Y
    Liu, F
    Insulin-stimulated activation of phosphoinositide-dependent Kinase-1 (PDK1): potential role in translocation of CLUT4 in rat adipose cells.2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 30, p. 11851-11859Article in journal (Refereed)
  • 7.
    Chi, Celestine N.
    et al.
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Haq, S. Raza
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Rinaldo, Serena
    University of Rome, Italy.
    Dogan, Jakob
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Cutruzzolà, Francesca
    University of Rome, Italy.
    Engström, Åke
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Gianni, Stefano
    University of Rome, Italy.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jemth, Per
    Department of Medical Biochemistry and Microbiology, Uppsala University.
    Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 44, p. 8971-8979Article in journal (Refereed)
    Abstract [en]

    The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

  • 8.
    Edvardsson, Anna
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Shapiguzov, Alexey
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Petersson, Ulrika A
    Schröder, Wolfgang P
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-22007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 33, p. 9432-9442Article in journal (Refereed)
    Abstract [en]

    The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates. © 2007 American Chemical Society.

  • 9.
    Fristedt, Rikard
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Carlberg, Inger
    Department of Biochemistry and Biophysics, Stockholm UniVersity, Stockholm, Sweden.
    Zygadlo, Agnieszka
    Department of Plant Biology, UniVersity of Copenhagen, Copenhagen, Denmark.
    Piippo, Mirva
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Nurmi, Markus
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Aro, Eva-Mari
    Department of Biology, Physiology and Molecular Biology, UniVersity of Turku, Turku, Finland.
    Vibe Scheller, Henrik
    Department of Plant Biology, UniVersity of Copenhagen, Copenhagen, Denmark.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Intrinsically Unstructured Phosphoprotein TSP9 Regulates Light Harvesting in Arabidopsis thaliana2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 2, p. 499-509Article in journal (Refereed)
    Abstract [en]

    Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

  • 10.
    Gréen, Anna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Sarg, Bettina
    DiVision of Clinical Biochemistry, Biocenter, Innsbruck Medical UniVersity, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
    Koutzamani, Elisavet
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Genheden, Ulrika
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Lindner, Herbert H.
    DiVision of Clinical Biochemistry, Biocenter, Innsbruck Medical UniVersity, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
    Rundquist, Ingemar
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, p. 7539-7547Article in journal (Refereed)
    Abstract [en]

    Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have been linked to the apoptotic process. Such conflicting results may depend on which various apoptosis-inducing agents cause apoptosis via different apoptotic pathways and often interfere with cell proliferation. Therefore, we investigated the relation between apoptosis and H1 phosphorylation in Jurkat cells after apoptosis induction via both the extrinsic and intrinsic pathways and by taking cell cycle effects into account. After apoptosis induction by anti-Fas, no significant dephosphorylation, as measured by capillary electrophoresis, or cell cycle-specific effects were detected. In contrast, H1 subtypes were rapidly dephosphorylated when apoptosis was induced by camptothecin. We conclude that histone H1 dephosphorylation is not connected to apoptosis in general but may be coupled to apoptosis by the intrinsic pathway or to concomitant growth inhibitory signaling.

  • 11.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Sekijima, Y.
    White, J.T.
    Costello, C.E.
    Altland, K.
    Garzuly, F.
    Budka, H.
    Kelly, J.W.
    D18G Transthyretin is Monomeric, Aggregation Prone, and Non-Detectable in Plasma and Cerbrospinal Fluid - A Prescription for CNS Amyloidosis?2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, p. 6656-6663Article in journal (Refereed)
  • 12.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Sekijima, Y.
    Skaggs Institute of Chemical Biology, Department of Chemistry, Scripps Research Institute, 10550 North Torrey Pines Road, San Diego, CA 92037, United States.
    White, J.T.
    Skaggs Institute of Chemical Biology, Department of Chemistry, Scripps Research Institute, 10550 North Torrey Pines Road, San Diego, CA 92037, United States.
    Wiseman, R.L.
    Skaggs Institute of Chemical Biology, Department of Chemistry, Scripps Research Institute, 10550 North Torrey Pines Road, San Diego, CA 92037, United States.
    Lim, A.
    Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118, United States.
    Costello, C.E.
    Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118, United States.
    Altland, K.
    Institut für Humangenetik, Justus-Liebigs-Universität, D-35392 Giessen, Germany.
    Garzuly, F.
    Department of Neurology, Markusovszky Hospital, Szombathely, Hungary.
    Budka, H.
    Institute of Neurology, University of Vienna, Vienna, Austria.
    Kelly, J.W.
    Skaggs Institute of Chemical Biology, Department of Chemistry, Scripps Research Institute, 10550 North Torrey Pines Road, San Diego, CA 92037, United States.
    D18G transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid: A prescription for central nervous system amyloidosis?2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, no 22, p. 6656-6663Article in journal (Refereed)
    Abstract [en]

    Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation, however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T4) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T4 CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.

  • 13.
    Hennig, Janosch
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. Helmholtz Zentrum Munchen GmbH, Germany; Technical University of Munich, Germany.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Museth, Anna Katrine
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology. CALTECH, CA 91125 USA.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Local Destabilization of the Metal-Binding Region in Human Copper-Zinc Superoxide Dismutase by Remote Mutations Is a Possible Determinant for Progression of ALS2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 2, p. 323-333Article in journal (Refereed)
    Abstract [en]

    More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 degrees C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 degrees C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer beta-strands I and VI of the beta-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 degrees C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local beta-strands.

  • 14.
    Hosia, W.
    et al.
    Dept. of Med. Biochem. and Biophys., Karolinska Institutet, S-171 77 Stockholm, Sweden.
    Bark, N.
    Dept. of Med. Biochem. and Biophys., Karolinska Institutet, S-171 77 Stockholm, Sweden, Department of Clinical Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden.
    Liepinsh, E.
    Dept. of Med. Biochem. and Biophys., Karolinska Institutet, S-171 77 Stockholm, Sweden.
    Tjernberg, A.
    Dept. of Med. Biochem. and Biophys., Karolinska Institutet, S-171 77 Stockholm, Sweden, Department of Structural Chemistry, Biovitrum AB, S-112 76 Stockholm, Sweden.
    Persson, Bengt
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics .
    Hallen, D.
    Hallén, D., Department of Structural Chemistry, Biovitrum AB, S-112 76 Stockholm, Sweden.
    Thyberg, J.
    Dept. of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
    Johansson, J.
    Dept. of Med. Biochem. and Biophys., Karolinska Institutet, S-171 77 Stockholm, Sweden, Department of Molecular Biosciences, Swed. Univ. of Agricultural Sciences, Biomedical Center, S-751 23 Uppsala, Sweden.
    Tjernberg, L.
    Department of Neurotec, Karolinska Institutet, S-141 57 Huddinge, Sweden.
    Folding into a ß-Hairpin Can Prevent Amyloid Fibril-Formation2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 16, p. 4655-4661Article in journal (Refereed)
    Abstract [en]

    The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable ß-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.

  • 15. Huang, S.
    et al.
    Sjoblom, B.
    Sauer-Eriksson, A.E.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Organization of an efficient carbonic anhydrase: Implications for the mechanism based on structure-function studies of a T199P/C206S mutant2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 24, p. 7628-7635Article in journal (Refereed)
    Abstract [en]

    Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)1 reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and ß-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The ß-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

  • 16.
    Hug, H.
    et al.
    Universitäts-Kinderklinik Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany, Universität Münster, Röntgen-Strasse 21, D-48149 Münster, Germany, and Orpegen Pharma, Czerny-Ring 22, D-69115 Heidelberg, Germany .
    Los, Marek Jan
    Department of Immunology and Cell Biology, University of Münster, Münster, Germany.
    Hirt, W.
    Orpegen Pharma, Czerny-Ring 22, D-69115 Heidelberg, Germany .
    Debatin, K. M.
    Universitäts-Kinderklinik Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany, Universität Münster, Röntgen-Strasse 21, D-48149 Münster, Germany.
    Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 42, p. 13906-13911Article in journal (Refereed)
    Abstract [en]

    Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is based on a substrate that contains only aspartate residues linked to rhodamine 110. With this and similar substrates, we are able to detect intracellular caspase activation by flow cytometry after apoptosis induction in intact hematopoetic cell lines.

  • 17.
    Kanmert, Daniel
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Brorsson, Ann-Christin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Enander, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Physics. Linköping University, Faculty of Science & Engineering.
    Thermal Induction of an Alternatively Folded State in Human IgG-Fc2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 6, p. 981-988Article in journal (Refereed)
    Abstract [en]

    We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

  • 18.
    Karlsson, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Carin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 9, p. 3487-3493Article in journal (Refereed)
    Abstract [en]

    The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).

  • 19.
    Karlsson, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Olofsson, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 21, p. 6803-6807Article in journal (Refereed)
    Abstract [en]

    The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond, the oxidized A23C/L203C mutant. This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium, molten-globule intermediate, which is normally very prone to aggregation. Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding. This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and, more importantly, to significantly higher reactivation yields upon refolding of the fully denatured protein. Thus, a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates, thereby shifting the unfolding behavior from a three-state process to a two-state one.

  • 20.
    Kjellsson, A.
    et al.
    Department of Biochemistry, Umeå University, SE-901 87 Umeå, Sweden.
    Sethson, I.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, no 2, p. 363-374Article in journal (Refereed)
    Abstract [en]

    The nature of denatured ensembles of the enzyme human carbonic anhydrase (HCA) has been extensively studied by various methods in the past. The protein constitutes an interesting model for folding studies that does not unfold by a simple two-state transition, instead a molten globule intermediate is highly populated at 1.5 M GuHCl. In this work, NMR and H/D exchange studies have been conducted on one of the isozymes, HCA I. The H/D exchange studies, which were enabled by the previously obtained resonance assignment of HCA I, have been used to identify unfolded forms that are accessible from the native state. In addition, the GuHCl-induced unfolded states of HCA I have also been characterized by NMR at GuHCl concentrations in the 0-5 M range. The most important findings in this work are as follows: (1) Amide protons located in the center of the ß-sheet require global unfolding events for efficient H/D exchange. (2) The molten globule and the native state give similar protection against H/D exchange for all of the observable amide protons (i.e., water seems not to efficiently penetrate the interior of the molten globule). (3) At high protein concentrations, the molten globule can form large aggregates, which are not detectable by solution-state NMR methods. (4) The unfolded state (U), present at GuHCl concentrations above 2 M, is composed of an ensemble of conformations having residual structures with different stabilities.

  • 21.
    Kukic, Predrag
    et al.
    University of Cambridge, England.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Camilloni, Carlo
    University of Cambridge, England.
    Evenas, Johan
    Red Glead Discovery, Sweden.
    Akke, Mikael
    Lund University, Sweden.
    Vendruscolo, Michele
    University of Cambridge, England.
    Structural Insights into the Calcium-Mediated Allosteric Transition in the C-Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 1, p. 19-28Article in journal (Refereed)
    Abstract [en]

    Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca2+-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, N-15 order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca2+ by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca2+-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations.

  • 22.
    Los, Marek Jan
    et al.
    Department of Immunology and Cell Biology, University of Münster, Münster, and German Cancer Research Center, Division of Molecular Genome Analysis, Heidelberg, Germany .
    Neubuser, D.
    Department of Immunology and Cell Biology, University of Münster, Münster, and German Cancer Research Center, Division of Molecular Genome Analysis, Heidelberg, Germany .
    Coy, J. F.
    German Cancer Research Center, Division of Molecular Genome Analysis, Heidelberg, Germany .
    Mozoluk, M.
    Department of Immunology and Cell Biology, University of Münster, Münster.
    Poustka, A.
    German Cancer Research Center, Division of Molecular Genome Analysis, Heidelberg, Germany .
    Schulze-Osthoff, Klaus
    Department of Immunology and Cell Biology, University of Münster, Münster, Germany .
    Functional characterization of DNase X, a novel endonuclease expressed in muscle cells2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 25, p. 7365-7373Article in journal (Refereed)
    Abstract [en]

    The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca2+ and Mg2+ and inhibited by Zn2+ ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.

  • 23.
    Lundqvist, Martin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Sethson, Ingmar
    Department of Organic Chemistry, Umeå University, Umeå, Sweden.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 30, p. 10093-10099Article in journal (Refereed)
    Abstract [en]

    It is well-known that adsorption of proteins on interfaces often induces substantial alterations of the protein structure. However, very little is known about whether these conformational changes have any consequence for the protein conformation after desorption from the interface. To investigate this matter, we have selected a protein−particle system in which the enzyme human carbonic anhydrase I (HCAI) alternates between the adsorbed and free state upon interaction with the silica nanoparticles. High-resolution NMR analysis of the protein with the particles present in the sample shows a spectrum that indicates a molten globular-like structure. Removal of particles results in refolding of virtually all HCAI molecules to a fully active form. However, the two-dimensional NMR analysis shows that refolding does not result in a single well-defined protein structure but rather provides an ensemble of protein molecules with near-native conformations. A detailed comparative chemical shift analysis of 108 amide signals in 1H−15N HSQC spectra of native and desorbed HCAI reveals that the most profound effects are located at β-strands in the center of the molecule. The observation of very slow H−D exchange in the central β-strands of HCAI [Kjellsson, A., Sethson, I., and Jonsson, B. H. (2003) Biochemistry 42, 363−374] in conjunction with our results indicates that the kinetic barriers for conformational rearrangements in the central core of the protein are low in the presence of nanoparticles but are very high under native conditions.

  • 24.
    Moparthi, Satish Babu
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Fristedt, Rikard
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mishra, Rajesh
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Almstedt, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 6, p. 1137-1145Article in journal (Refereed)
    Abstract [en]

    The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgård, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466−468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

  • 25.
    Mrosek, Michael
    et al.
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Meier, Sebastian
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Ucurum-Fotiadis, Zöhre
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    von Castelmur, Eleonore
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Hedbom, Erik
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Lustig, Ariel
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Grzesiek, Stephan
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland.
    Labeit, Dietmar
    Institut für Anästhesiologie and Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim, Germany.
    Labeit, Siegfried
    Institut für Anästhesiologie and Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim, Germany.
    Mayans, Olga
    Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland; School of Biological Sciences, University of Liverpool, Crown Street, Liverpool, U.K..
    Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold.2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 40, p. 10722-10730Article in journal (Refereed)
    Abstract [en]

    The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.

  • 26.
    Mårtensson, Lars-Göran
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, p. 15867-15875Article in journal (Refereed)
    Abstract [en]

    To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten−globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

  • 27.
    Nilsson, Peter
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Herland, Anna
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 10, p. 3718-3724Article in journal (Refereed)
    Abstract [en]

    The in vivo deposition of amyloid fibrils is a hallmark of many devastating diseases known as the amyloidoses. Amyloid formation in vitro may also complicate production of proteins in the biotechnology industry. Simple, sensitive, and versatile tools that detect the fibrillar conformation of amyloidogenic proteins are thus of great importance. We have developed a negatively charged conjugated polyelectrolyte that displays different characteristic optical changes, detected visually or by absorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. This simple, rapid, and novel methodology was applied here to two amyloidogenic proteins, insulin and lysozyme, and its validity for detection of their fibrillar conformation was verified by currently used methods such as circular dichroism, transmission electron microscopy, and Congo red absorption.

  • 28.
    Osterlund, Maria
    et al.
    Novo Nordisk AS, Prot Biotechnol, DK-2880 Bagsvaerd, Denmark Linkoping Univ, IFM, Dept Chem, Linkoping, Sweden Linkoping Univ, IFM, Dept Phys Chem, Linkoping, Sweden Novo Nordisk AS, Vasc Biochem, Malov, Denmark.
    Owenius, Rikard
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Carlsson, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Egon
    Vascular Biochemistry, Novo Nordisk A/S, Denmark.
    Lindgren, Mikael
    Department of Laser Systems, Division of Sensor Technology, Swedish Defence Research Agency, P.O. Box 1165, SE- 581 11 Linko¨ping, Sweden..
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 31, p. 9324-9328Article in journal (Refereed)
    Abstract [en]

    Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

  • 29.
    Persson, Malin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    EPR Mapping of Interactions Between Spin-labeled Variants of Human Carbonic Anhydrase II and GroEL. Evidence for increased flexibility of the hydrophobic core by the interaction.1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, p. 432-441Article in journal (Refereed)
  • 30.
    Ruiz Pavón, Lorena
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Karlsson, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Samyn, Dieter
    School of Pure and Applied Natural Sciences, Kalmar University, 391 82 Kalmar, Sweden.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Persson, Bengt L.
    School of Pure and Applied Natural Sciences, Kalmar University, 391 82 Kalmar, Sweden.
    Spetea, Cornelia
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-directed Mutagenesis2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 30, p. 6430-6439Article in journal (Other academic)
    Abstract [en]

    The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian SLC17 family, has recently been localized to the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acids involved in substrate binding/translocation by ANTR1 and in the Na+-dependence of its activity. A threedimensional structural model of ANTR1 was constructed using the crystal structure of glycerol-3-phosphate/phosphate antiporter (GlpT) from E.coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely Arg-120, Ser-124 and Arg-201 inside the putative translocation pathway, Arg-228 and Asp-382 exposed at the cytosolic surface of the protein. The activities of the wild type and mutant proteins have been analyzed using expression in E. coli and radioactive transport assays, and compared with bacterial cells carrying an empty plasmid. Based on Pi- and Na+-dependent kinetics, we propose that Arg-120, Arg-201 and Arg-228 are involved in binding and translocation of the substrate, Ser-124 functions as a periplasmic gate for Na+ ions, and finally Asp-382 participates in the turnover of the transporter via ionic interaction with either Arg-228 or Na+ ions. We also propose that the corresponding residues may have a similar function in other plant and mammalian SLC17 homologous transporters.

  • 31.
    Shutova, T
    et al.
    Umeå University.
    Nikitina, J
    Umeå University.
    Deikus, G
    New York University.
    Andersson, Bertil
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Klimov, V
    Russian Academy of Sciences.
    Samuelsson, G
    Umeå University.
    Structural dynamics of the manganese-stabilizing protein - Effect of pH, calcium, and manganese2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 46, p. 15182-15192Article in journal (Refereed)
    Abstract [en]

    The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.2, the hydrophobic amino acid residues that are most likely located inside the beta barrel are "closed" and the protein binds neither Mn2+ nor Ca2+ ions. When the protein is transferred to a solution with a slightly acidic pH of 5.7, hydrophobic amino acid residues become exposed to the surrounding medium, enabling them to bind the fluorescent probe 8,1-ANS. At this pH-induced open state, Mn2+ and Ca2+ bind to the manganese-stabilizing protein. The pH values used in this study, 7.2 and 5.7, are typical of the pH found in the thylakoid lumen in the dark and light, respectively. A model is presented in which the manganese-stabilizing protein undergoes a pH-dependent conformational change that in turn influences its capacity to bind calcium and manganese. In this model, the proton-dependent conformational changes of the tertiary structure of the manganese-stabilizing protein are of functional relevance for the regulation of substrate (water) delivery to and product (proton) release from the water-oxidizing complex by forming a proton-sensing proton-transport pathway

  • 32. Song, Jikiu
    et al.
    Lee, Min S
    Carlberg, Inger
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Markley, John L
    Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 51, p. 15633-15643Article in journal (Refereed)
    Abstract [en]

    Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal α-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal α-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function. © 2006 American Chemical Society.

  • 33. Stenlund, Peter
    et al.
    Lindberg, Mikael J
    Tibell, Lena
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Structural requirements for high-affinity heparin binding: Alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 9, p. 3168-3175Article in journal (Refereed)
    Abstract [en]

    An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.

  • 34.
    Tibell, Lena
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Lookene, Aivar
    Medical chemistry Umeå.
    Stenlund, Peter
    Biochemistry Umeå.
    Characterization of the Heparin Binding of Human Extracellular Superoxide Dismutase2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, p. 230-236Article in journal (Refereed)
    Abstract [en]

     The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, Kd, of 0.12 M, which is 700 and 10-20 times lower than the Kd values for the synthetic peptide and the fusion proteins, respectively. However, when an -helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the Kd decreased to 0.64 M. The differences in the Kd values were mainly governed by differences in the association rate constants (kass). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s-1), and a very high association rate constant (8 × 105 M-1 s-1), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an -helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.

  • 35.
    Villebeck, Laila
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Moparthi, Satish Babu
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lindgren, Mikael
    Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Domain-specific chaperone-induced expansion is required for ß-actin folding: a comparison of ß-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 44, p. 12639-12647Article in journal (Refereed)
    Abstract [en]

    Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native ß-actin, but is unable to guide ß-actin toward the native state. In this study we identify conformational rearrangements in ß-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of ß-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled ß-actin was characterized, and the structural rearrangement was compared to the case of the ß-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the ß-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound ß-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the ß-actin molecule. It can be concluded that the specific and directed rearrangement of the ß-actin structure, seen in the natural ß-actin-TRiC system, is vital for guiding ß-actin to the native state. © 2007 American Chemical Society.

  • 36. Vince, JW
    et al.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Reithmeier, RAF
    Localization of the Cl-/HCO3- anion exchanger binding site to the amino-terminal region of carbonic anhydrase II2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 44, p. 13344-13349Article in journal (Refereed)
    Abstract [en]

    Human carbonic anhydrase II (CAII) possesses a binding site for an acidic motif (D887ADD) within the carboxyl-terminal region (Ct) of the human erythrocyte chloride/bicarbonate anion exchanger, AE1. In this study, the amino acid sequence comprising this AE1 binding site was localized to the first 17 residues of CAII, which form a basic patch on the surface of the protein. Truncation of the amino terminal of CAII by five residues resulted in a 3-fold reduction in the apparent affinity of the interaction with a GST fusion protein of the Ct of AE1 (GST-Ct) measured by a sensitive microtiter plate binding assay. Further amino-terminal truncation of CAII by 17 or 24 residues caused a loss of binding. The homologous isoform CAI does not bind AE1, despite having 60% sequence identity to CAII. One major difference between the two CA isoforms, within the amino-terminal region, is a high content of histidine residues in CAII (His3, -4, -10, -15, -17) not found in CAI. Mutation of pairs of these histidines (and one lysine) in CAII to the analogous residues in CAI (H3P/H4D or K9D/H10K or H15Q/H17S), or combinations of these various double mutants, did not greatly affect binding between GST-Ct and the mutant CAII. However, when all six of the targeted CAII residues were mutated to the corresponding sequence in CAI, binding of GST-Ct was lost. These results indicate that the AE1 binding site is located within the first 17 residues of CAII, and that the interaction is mediated by electrostatic interactions involving histidine and/or lysine residues. Further specificity for the interaction of AE1 and CAII is provided by a conserved leucine residue (L886) in AE1 that, when mutated to alanine, resulted in loss of GST-Ct binding to immobilized CAII. The binding of the basic amino-terminal region of CAII to an acidic Ct in AE1 provides a structural basis for linking bicarbonate transport across the cell membrane to intracellular bicarbonate metabolism.

  • 37. Weber, A
    et al.
    Dalen, Helge
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Andera, L
    Nègre-Salvayre, A
    Augé, N
    Sticha, M
    Loret, A
    Terman, Alexei
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Witting, PK
    Higuchi, M
    Plasilova, M
    Zivny, J
    Gellert, N
    Weber, C
    Neuzil, Jiri
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Mitochondria play a central role in apoptosis induced by a-tocopheryl succinate, an agent with antineoplastic activity: Comparison with receptor-mediated pro-apoptotic signaling2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, no 14, p. 4277-4291Article in journal (Refereed)
    Abstract [en]

    a-Tocopheryl succinate (a-TOS) is a semisynthetic vitamin E analogue with high pro-apoptotic and anti-neoplastic activity [Weber, T et al. (2002) Clin. Cancer Res. 8, 863-869]. Previous studies suggested that it acts through destabilization of subcellular organelles, including mitochondria, but compelling evidence is missing. Cells treated with a-TOS showed altered mitochondrial structure, generation of free radicals, activation of the sphingomyelin cycle, relocalization of cytochrome c and Smac/Diablo, and activation of multiple caspases. A pan-caspase inhibitor suppressed caspase-3 and -6 activation and phosphatidyl serine externalization, but not decrease of mitochondrial membrane potential or generation of radicals. For a-TOS, but not Fas or TRAIL, apoptosis was suppressed by caspase-9 inhibition, while TRAIL- and Fas-resistant cells overexpressing cFLIP or CrmA were susceptible to a-TOS. The central role of mitochondria was confirmed by resistance of mtDNA-deficient cells to a-TOS, by regulation of a-TOS apoptosis by Bcl-2 family members, and by anti-apoptotic activity of mitochondrially targeted radical scavengers. Co-treatment with a-TOS and anti-Fas IgM showed their cooperative effect, probably by signaling via different, convergent pathways. These data provide an insight into the molecular mechanism, by which a-TOS kills malignant cells, and advocate its testing as a potential anticancer agent or adjuvant.

  • 38.
    Wennerstrand, Patricia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Dametto, Paolo
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Klingstedt, Therése
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*52012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 30, p. 5912-5920Article in journal (Refereed)
    Abstract [en]

    The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

  • 39. Wirehn, J.
    et al.
    Carlsson, Karin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Herland, Anna
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics .
    Persson, E.
    Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Svensson, Magdalena
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 18, p. 6755-6763Article in journal (Refereed)
    Abstract [en]

    Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gavériaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTFR200W) compared to that of the wild-type protein (sTF wt). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTFR200W mutant and sTFwt were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTFR200W indicated a population of partially folded intermediates during denaturation. sTFR200W showed a dramatically increased propensity for aggregate formation compared to sTFwt at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTFR200W in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TFR200W carriers, which could protect from thrombotic disease. © 2005 American Chemical Society.

  • 40.
    Zer, H.
    et al.
    Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
    Vink, M.
    Department of Biochemistry, Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden.
    Shochat, S.
    Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
    Herrmann, R.G.
    Institute of Botany, Ludwig Maximilians University, D-80638 Münich, Germany.
    Andersson, B.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Ohad, I.
    Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
    Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)2003In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, no 3, p. 728-738Article in journal (Refereed)
    Abstract [en]

    Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30, µmol m-2 s-1) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 µmol m-2 s-1) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5°C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.

  • 41. Zyryanov, AB
    et al.
    Vener, Alexander
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Salminen, A
    Goldman, A
    Lahti, R
    Baykov, AA
    Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 4, p. 1065-1074Article in journal (Refereed)
    Abstract [en]

    Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn2+ rather than Mg2+, the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and 11 PPases. We measured rates of PPi hydrolysis, PPi synthesis, and P-i/water oxygen exchange catalyzed by sgPPase with Mn2+, Mg2+, or Co2+ in the high-affinity metal-binding site and Mg2+ in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P-i). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PPi complex isomerization known to occur in family I PPases, (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PPi hydrolysis and product release, (c) the marked acceleration of sgPPase catalysis in the presence of Mn2+ and Co2+ results from a combined effect of these ions on bound PPi hydrolysis and P-i release, (d) sgPPase exhibits lower affinity for both PPi and P-i, and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K-m, which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PPi concentrations).

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