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  • 1. Alam, MT
    et al.
    Yamada, T
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Ikai, A
    The importance of being knotted: Effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II2003In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 84, no 2, p. 159A-159AConference paper (Other academic)
  • 2.
    Alam, M.T.
    et al.
    Laboratory of Biodynamics, Grad. Sch. of Biosci. and Biotech., Tokyo Inst. of Technol., 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
    Yamada, T.
    Laboratory of Biodynamics, Grad. Sch. of Biosci. and Biotech., Tokyo Inst. of Technol., 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Ikai, A.
    Laboratory of Biodynamics, Grad. Sch. of Biosci. and Biotech., Tokyo Inst. of Technol., 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
    The importance of being knotted: Effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II12002In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 519, no 1-3, p. 35-40Article in journal (Refereed)
    Abstract [en]

    In order to better understand the contribution of the knotted folding pattern to the enzymatic and mechanical properties of carbonic anhydrases, we replaced Gln-253 of bovine carbonic anhydrase II with Cys, which allowed us to measure the mechanical strength of the protein against tensile deformation by avoiding knot tightening. The expressed protein, to our surprise, turned out to contain two conformational isomers, one capable of binding an enzymatic inhibitor and the other not, which led to their separation through affinity chromatography. In near- and far-UV circular dichroism and fluorescence spectra, the separated conformers were very similar to each other and to the wild-type enzyme, indicating that they both had native-like conformations. We describe new evidence which supports the notion that the difference between the two conformers is likely to be related to the completeness of the C-terminal knot formation. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

  • 3.
    Almstedt, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Protein Misfolding in Human Diseases2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:

    1. Loss-of-function diseases
    2. Gain-of-toxic-function diseases
    3. Infectious misfolding diseases

     

    Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. 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 towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.

     

    The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.

    Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

    List of papers
    1. Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
    Open this publication in new window or tab >>Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
    Show others...
    2004 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article in journal (Refereed) Published
    Abstract [en]

    Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

    Place, publisher, year, edition, pages
    Oxford: Elsevier, 2004
    Keywords
    Misfolding, loss-of-function, aggregation, molten globule, misfolding inhibitor
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:liu:diva-21072 (URN)10.1016/j.jmb.2004.07.024 (DOI)
    Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
    2. Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
    Open this publication in new window or tab >>Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
    2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    Washington: ACS, 2008
    Keywords
    mutant mapping, carbonic anhydrase, misfolding, loss-of-function, molten globule
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:liu:diva-21056 (URN)10.1021/bi701720p (DOI)
    Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
    3. Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
    Open this publication in new window or tab >>Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
    Show others...
    2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 23, p. 5358-5364Article in journal (Refereed) Published
    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.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-19548 (URN)10.1021/bi900128e (DOI)
    Note
    On the day of the defence date tha status of this articel was In Manuscript.Available from: 2009-06-29 Created: 2009-06-26 Last updated: 2018-04-25Bibliographically approved
    4. Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
    Open this publication in new window or tab >>Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
    2009 (English)In: Prion, ISSN 1933-6896, Vol. 3, no 4, p. 224-235Article in journal (Refereed) Published
    Abstract [en]

    Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.

    Place, publisher, year, edition, pages
    Austin: Landes Bioscience Journals, 2009
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-21064 (URN)10.4161/pri.3.4.10112 (DOI)000280061100009 ()
    Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25
  • 4.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Lundqvist, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Carlsson, Jonas
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
    Karlsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Bengt
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . 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.
    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.
    Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article in journal (Refereed)
    Abstract [en]

    Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

  • 5.
    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.

  • 6.
    Almstedt, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates2009In: Prion, ISSN 1933-6896, Vol. 3, no 4, p. 224-235Article in journal (Refereed)
    Abstract [en]

    Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.

  • 7.
    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.

  • 8. 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.

  • 9.
    Andersson, Dick
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor: Application in folding studies and prediction of secondary structure2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 4, p. 1118-1128Article in journal (Refereed)
    Abstract [en]

    The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180-305 nm). The sum of the individual spectra of each Trp residue in the near-UV region was qualitatively identical to the wild-type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far-UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum obtained after subtraction of all individual Trp spectra from the wild-type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native-like tertiary structure by kinetic near-UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side-chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.

  • 10.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Jalal, Shah
    Karolinska University Hospital.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Wang, Yi
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Islam, Sohidul
    Karolinska University Hospital.
    Jarl, Anngelica
    Linköping University, Department of Physics, Chemistry and Biology, Molecular genetics . Linköping University, The Institute of Technology.
    Liedberg, Bo
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics . Linköping University, The Institute of Technology.
    Wretlind, Bengt
    Karolinska University Hospital.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance2010In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 4, p. 680-692Article in journal (Refereed)
    Abstract [en]

    The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

  • 11.
    Berg, Ina
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Modeling Amyloid Disease in Drosophila melanogaster2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid diseases are caused by protein misfolding and aggregation. To date there are 27 known proteins causing amyloid disorders involving brain and peripheral protein deposition. The proteins involved in this mechanism do not share sequence homology, but the amyloid fibrils share biophysical properties and possibly a common pathogenic mechanism. Amyloid deposits are known to be involved in a broad range of neurodegenerative diseases, such as Alzheimer’s disease and Creutzfeldt-Jakob disease, as well as in non-neuropathic diseases, such as senile systemic amyloidosis and type II diabetes.

    During the last decade the fruit fly, Drosophila melanogaster (Drosophila), have increasingly been used as a model for neurodegenerative disease, such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and familial amyloidotic polyneuropathy. The advantages of using the Drosophila model are the well-defined genetic characteristics, the quantity, short life span, simplicity in genetic manipulation and the powerful binary UAS-Gal4 transgenic system. The UAS-Gal4 system allows for rapid generation of individual strains in which expression of a specific gene of interest can be directed to different tissues or cell types. The system allows the target gene to be activated in different cell- and tissue-types by altering the activator-expressing lines.

    This thesis has been focused on modeling amyloid diseases in Drosophila. This has been performed by:

    • Creating new model systems of senile systemic amyloidosis and familial amyloidotic polyneuropathy in Drosophila
    • Developing a new staining protocol for detection of amyloid in Drosophila
    • Initiate a compound screen of Alzheimer’s disease modeled in Drosophila
    List of papers
    1. Modeling Familial Amyloidotic Polyneuropathy (Transthyretin V30M) in Drosophila melanogaster
    Open this publication in new window or tab >>Modeling Familial Amyloidotic Polyneuropathy (Transthyretin V30M) in Drosophila melanogaster
    2009 (English)In: NEURODEGENERATIVE DISEASES, ISSN 1660-2854, Vol. 6, no 3, p. 127-138Article in journal (Refereed) Published
    Abstract [en]

    Background/Aims: Transthyretin (TTR) is a prevalent plasma and cerebrospinal fluid protein associated with sporadic and heritable amyloidosis. TTR amyloidosis is linked to a vast number of mutations with varying phenotype, tissue distribution and age of onset. The most prevalent mutation associated with familial amyloidotic polyneuropathy (FAP) is the V30M mutation. Studies of transgenic mouse models of TTR V30M FAP have been hampered by variable phenotype, low disease penetrance, and slow onset. Methods/Results: To model TTR-associated amyloid disease in the Drosophila model system, transgenic Drosophila were generated, expressing wild-type (wt) TTR or TTR V30M, associated with sporadic senile systemic amyloidosis (SSA) and inherited FAP, respectively. We found that expression of FAP-associated TTR V30M mutant in the nervous system resulted in reduced lifespan and in reduced climbing ability indicating neurological impairment, whereas expression of TTR wt showed a milder phenotype. Congo red staining of the Drosophila brain shows positive amyloid binding in the aged TTR V30M flies. Extensive brain vacuole formation was evident for the aged TTR V30M flies, whereas a milder phenotype was shown by the TTR wt flies. In addition, expression of TTR V30M in the eye leads to tissue damage, including rough eye, morphological changes and fibrous deposition. Conclusion: Our results suggest that Drosophila is a promising complementary system for studies of TTR-associated amyloid diseases.

    Keywords
    Amyloid disease, Drosophila melanogaster, Transthyretin, Familial amyloidotic polyneuropathy
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-18382 (URN)10.1159/000213761 (DOI)
    Available from: 2009-05-25 Created: 2009-05-25 Last updated: 2018-04-25
    2. Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses
    Open this publication in new window or tab >>Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses
    2010 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 5, no 5, p. 935-944Article in journal (Refereed) Published
    Abstract [en]

    Drosophila melanogaster is emerging as an important model system for neurodegenerative disease research. In this protocol, we describe an efficient method for imaging amyloid deposits in the Drosophila brain, by the use of a luminescent-conjugated oligothiophene (lco), p-Ftaa polymer probe. We also demonstrate the feasibility of co-staining with antibodies and compare the lco staining with standard amyloid-specific probes. the lco protocol enables high-resolution imaging of several different protein aggregates, such as aβ1-42, aβ1-42e22G, transthyretin V30M and human tau, in the Drosophila brain. aβ and tau aggregates could also be distinguished from each other because of distinct lco emission spectra. Furthermore, this protocol enables threedimensional brain mapping of amyloid distribution in whole-mount Drosophila brains. the use of p-Ftaa combined with other probes, antibodies and/or dyes will aid the rapid characterization of various amyloid deposits in the rapidly growing number of Drosophila models of neurodegenerative diseases.

    Place, publisher, year, edition, pages
    Macmillan Publishers Ltd., 2010
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-55024 (URN)10.1038/nprot.2010.41 (DOI)000277174100012 ()
    Available from: 2010-04-27 Created: 2010-04-27 Last updated: 2018-04-25Bibliographically approved
    3. Curcumin alleviates Aβ indcuced neurotoxicity and vice versa without removing amyloid deposits in transgenic Drosophila
    Open this publication in new window or tab >>Curcumin alleviates Aβ indcuced neurotoxicity and vice versa without removing amyloid deposits in transgenic Drosophila
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Curcumin has been proposed to facilitate clearance of toxic amyloid formed by the Aβ peptide. To further address this notion, different concentrations of curcumin were tried for its effects in various Drosophila Alzheimer’s disease (AD) models. This study entailed five different Drosophila AD models (four Aβ expressing lines, and one tau expressing line), expressing the AD associated proteins using the Gal4/UAS system. These were assayed for several aspects of neurological impairment, including survival, climbing behavior, as well as locomotor activity. In addition, amyloid deposition was assessed by histological analysis. Curcumin treatment substantially prolonged the lifespan and improved climbing and locomotor activity for flies with severe disease geneotypes (Aβ1-42 E22G and double expressing Aβ1-42). In comparison, curcumin feeding of control flies resulted in a concentration-dependent shortened lifespan, whereas no such toxic side effects were found for AD genotypes with a mild phenotype (single expressors of Aβ1-40 and Aβ1-42). All flies expressing Aβ and tau displayed a higher total locomotor activity, and a continuation of the activity over a larger number of hours upon curcumin treatment. Unexpectedly, no change in tissue amyloid deposition upon curcumin treatment was observed. In vitro fibrillation of Aβ1-42, followed by Western blot and transmission electron microscopy in the presence and absence of curcumin, displayed enhanced fibrillation into large aggregates and decreased population of oligomers in curcumin samples. The decrease in oligomer formation by curcumin may explain why it increases the lifespan and activity without removing of the amyloid deposits seen in tissues. We also suggest that Aβ, at least in the context of Drosophila, functions as a chemical detoxifier sequestering curcumin and thereby mitigating its toxicity.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-55020 (URN)
    Available from: 2010-04-27 Created: 2010-04-27 Last updated: 2018-04-25
  • 12.
    Berg, Ina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Curcumin alleviates Aβ indcuced neurotoxicity and vice versa without removing amyloid deposits in transgenic DrosophilaManuscript (preprint) (Other academic)
    Abstract [en]

    Curcumin has been proposed to facilitate clearance of toxic amyloid formed by the Aβ peptide. To further address this notion, different concentrations of curcumin were tried for its effects in various Drosophila Alzheimer’s disease (AD) models. This study entailed five different Drosophila AD models (four Aβ expressing lines, and one tau expressing line), expressing the AD associated proteins using the Gal4/UAS system. These were assayed for several aspects of neurological impairment, including survival, climbing behavior, as well as locomotor activity. In addition, amyloid deposition was assessed by histological analysis. Curcumin treatment substantially prolonged the lifespan and improved climbing and locomotor activity for flies with severe disease geneotypes (Aβ1-42 E22G and double expressing Aβ1-42). In comparison, curcumin feeding of control flies resulted in a concentration-dependent shortened lifespan, whereas no such toxic side effects were found for AD genotypes with a mild phenotype (single expressors of Aβ1-40 and Aβ1-42). All flies expressing Aβ and tau displayed a higher total locomotor activity, and a continuation of the activity over a larger number of hours upon curcumin treatment. Unexpectedly, no change in tissue amyloid deposition upon curcumin treatment was observed. In vitro fibrillation of Aβ1-42, followed by Western blot and transmission electron microscopy in the presence and absence of curcumin, displayed enhanced fibrillation into large aggregates and decreased population of oligomers in curcumin samples. The decrease in oligomer formation by curcumin may explain why it increases the lifespan and activity without removing of the amyloid deposits seen in tissues. We also suggest that Aβ, at least in the context of Drosophila, functions as a chemical detoxifier sequestering curcumin and thereby mitigating its toxicity.

  • 13.
    Berg, Ina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses2010In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 5, no 5, p. 935-944Article in journal (Refereed)
    Abstract [en]

    Drosophila melanogaster is emerging as an important model system for neurodegenerative disease research. In this protocol, we describe an efficient method for imaging amyloid deposits in the Drosophila brain, by the use of a luminescent-conjugated oligothiophene (lco), p-Ftaa polymer probe. We also demonstrate the feasibility of co-staining with antibodies and compare the lco staining with standard amyloid-specific probes. the lco protocol enables high-resolution imaging of several different protein aggregates, such as aβ1-42, aβ1-42e22G, transthyretin V30M and human tau, in the Drosophila brain. aβ and tau aggregates could also be distinguished from each other because of distinct lco emission spectra. Furthermore, this protocol enables threedimensional brain mapping of amyloid distribution in whole-mount Drosophila brains. the use of p-Ftaa combined with other probes, antibodies and/or dyes will aid the rapid characterization of various amyloid deposits in the rapidly growing number of Drosophila models of neurodegenerative diseases.

  • 14.
    Berg, Ina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Thor, Stefan
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Modeling Familial Amyloidotic Polyneuropathy (Transthyretin V30M) in Drosophila melanogaster2009In: NEURODEGENERATIVE DISEASES, ISSN 1660-2854, Vol. 6, no 3, p. 127-138Article in journal (Refereed)
    Abstract [en]

    Background/Aims: Transthyretin (TTR) is a prevalent plasma and cerebrospinal fluid protein associated with sporadic and heritable amyloidosis. TTR amyloidosis is linked to a vast number of mutations with varying phenotype, tissue distribution and age of onset. The most prevalent mutation associated with familial amyloidotic polyneuropathy (FAP) is the V30M mutation. Studies of transgenic mouse models of TTR V30M FAP have been hampered by variable phenotype, low disease penetrance, and slow onset. Methods/Results: To model TTR-associated amyloid disease in the Drosophila model system, transgenic Drosophila were generated, expressing wild-type (wt) TTR or TTR V30M, associated with sporadic senile systemic amyloidosis (SSA) and inherited FAP, respectively. We found that expression of FAP-associated TTR V30M mutant in the nervous system resulted in reduced lifespan and in reduced climbing ability indicating neurological impairment, whereas expression of TTR wt showed a milder phenotype. Congo red staining of the Drosophila brain shows positive amyloid binding in the aged TTR V30M flies. Extensive brain vacuole formation was evident for the aged TTR V30M flies, whereas a milder phenotype was shown by the TTR wt flies. In addition, expression of TTR V30M in the eye leads to tissue damage, including rough eye, morphological changes and fibrous deposition. Conclusion: Our results suggest that Drosophila is a promising complementary system for studies of TTR-associated amyloid diseases.

  • 15.
    Birgersson, Elin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Gene identification in the encystation pathway of the Dictyostelid Polysphondylium pallidum2011Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Encystation of unicellular organisms is of considerable medical relevance since cysts are encapsulated byresilient cell walls, rendering them resistant to biocides and immune clearance. This survival strategymakes it complicated to produce effective treatment of diseases caused by many protozoan pathogens,e.g. species of Acanthamoeba which causes fatal granulomatous amebic encephalitis (GAE) and keratitis.Due to genetic limitations in most protists, the machinery of encystation is so far little understood.However, the signalling pathways can be investigated in the non-pathogenic social amoebas, Dictyostelia.In this master’s project, five genes in Polysphondylium pallidum were investigated, which might beinvolved in encystation. Research has demonstrated a relationship between encystation and the cyclicadenosine monophosphate (cAMP) signalling pathways in Dictyostelia spore formation. This indicates thatcysts are ancestral to spores, and hence are the sporulation genes: pkaC, yakA, regA, cudA and srfAselected for this study. The genes were individually knocked-out by a standard homologous recombinationapproach and the genes’ contribution to encystation was determined. Five knock-out constructs werecompleted and a preliminary analysis of the role of the intracellular cAMP phosphodiesterase RegA inPolysphondylium pallidum encystation process was performed.

  • 16.
    Carlsson, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Tissue Factor in Complex: Studies of interactions between blood coagulation proteins2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Many biological processes rely on specific protein-protein interactions, for example immune responses, cell signaling, transcription, and blood coagulation. Blood coagulation is initiated when a vessel wall is damaged, exposing tissue factor (TF) to the circulating factor VII/factor VIIa (FVII/FVIIa) which results in the formation of the TF:FVIIa complex and thereby the initiation of blood coagulation. One of the substrates for the TF:FVIIa complex is factor X (FX), which is activated to factor Xa (FXa), subsequently leading to a series of reactions resulting in clot formation. Tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of the sTF:FVIIa complex, involved in regulation of coagulation by forming the TF:FVIIa:FXa:TFPI complex. Occasionally, the blood coagulation mechanism malfunctions, resulting in conditions such as the inability to stop bleeding or thrombosis. The fact that TF is the main initiator of the coagulation makes this an interesting protein to study, in the hunt for means to interfere with players involved in the blood clotting process.

    Throughout the studies included in this thesis the site-directed labeling technique is utilized to attach spectroscopic probes to cysteines, introduced at specific positions by mutagenesis, in the protein of interest. These fluorescent or spin-probes are sensitive for changes in their immediate environment and can thus, for example be used to monitor protein-protein complex formation and conformational changes.

    No complete structure has been obtained as yet for the large complex involving sTF, FVIIa, FXa, and TFPI. Therefore, we introduced a fluorescent probe at specific positions in soluble tissue factor (sTF) and the changes in fluorescence emission were detected upon sTF:FVIIa:FXa:TFPI complex formation. From these measurements it was concluded that not only parts of the C-terminal domain of sTF (TF2), but also residues in the N-terminal domain (TF1) are involved in binding to FXa in the quaternary complex.

    In order to investigate conformational changes occurring in the extended interface between sTF and FVIIa upon binding of different inhibitors spectroscopic probes were introduced in sTF, in the vicinity of the interaction region. From the obtained data it was concluded that the exosite-binding inhibitor E-76 induces equivalent structural changes at the interface of sTF and the protease domain (PD) of FVIIa, as do the active-site inhibitors FFR and TFPI, i.e. makes the region around the active-site more compact. Binding of these inhibitors shows similar effects despite their differences in size, binding site, and inhibitory mechanism.

    In addition, the Ca2+ dependence of the formation of the sTF:FVIIa complex was studied. Association between sTF and FVIIa during Ca2+ titration begins by Ca2+ binding to the first EGF-like domain of FVIIa. However, Ca2+ saturation of the γ-carboxyglutamic acid-rich (Gla) domain of FVIIa is required for complete sTF:FVIIa complex formation, and we were also able to detect that a Gla domain with vacant Ca2+ sites hinders the docking to sTF.

    Finally, we investigated the structural changes of free inhibited FVIIa upon sTF and Ca2+ binding by FRET and quenching measurements. From this it was concluded that inhibited FVIIa does not seem to undergo large global structural changes upon binding to sTF, when taking the dynamics of free FVIIa into account. However, Ca2+ binding induces minor local conformational changes in the active-site region of the PD of inhibited FVIIa and subsequent binding of sTF causesfurther structural rearrangements in this area.

    List of papers
    1. Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor
    Open this publication in new window or tab >>Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor
    Show others...
    2003 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 12, p. 2576-2582Article in journal (Refereed) Published
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

    Keywords
    Fluorescence, Local probing, Protein-protein interactions, Site-directed labeling
    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-46604 (URN)10.1046/j.1432-1033.2003.03625.x (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
    2. Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa
    Open this publication in new window or tab >>Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa
    Show others...
    2001 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 31, p. 9324-9328Article in journal (Refereed) Published
    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.

    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-49196 (URN)10.1021/bi010283n (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
    3. Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor
    Open this publication in new window or tab >>Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor
    2006 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 349, no 3, p. 1111-1116Article in journal (Refereed) Published
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-36179 (URN)10.1016/j.bbrc.2006.08.148 (DOI)30411 (Local ID)30411 (Archive number)30411 (OAI)
    Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2018-03-23
    4. Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains
    Open this publication in new window or tab >>Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains
    Show others...
    2003 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1648, no 1-2, p. 12-16Article in journal (Refereed) Published
    Abstract [en]

    We have used the site-directed labeling approach to study the Ca 2+-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca2+ binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the ?-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca2+ titration is initiated upon Ca2+ binding to EGF1, the domain containing the site of highest Ca2+ affinity. Besides we showed that a Ca 2+-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca2+ binding to the PD seems to be of some importance for the docking of this domain to sTF. © 2003 Elsevier Science B.V. All rights reserved.

    Keywords
    Ca2+ dependence, Factor VIIa, Fluorescence, Protein-protein interaction, Tissue factor, Titration
    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-46620 (URN)10.1016/S1570-9639(03)00025-6 (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
    5. Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
    Open this publication in new window or tab >>Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
    Show others...
    2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, p. 545-549Article in journal (Refereed) Published
    Abstract [en]

    The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.

    Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

    Place, publisher, year, edition, pages
    Elsevier, 2011
    Keywords
    Factor VIIa, Fluorescence quenching, Fluorescence resonance energy transfer, Tissue factor, Fluorescein, Rhodamine, Conformational dynamics
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:liu:diva-63686 (URN)10.1016/j.bbrc.2011.08.135 (DOI)000295912800010 ()
    Note
    Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation||Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2017-12-11Bibliographically approved
  • 17.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Freskgard, P.-O.
    Freskgård, P.-O., Protein Biotechnology, Novo Nordisk A/S, Novo Allé, Bagsværd, Denmark, Nuevolution A/S, Rønnegade 8, Copenhagen, Denmark.
    Persson, E.
    Haemostasis Biology, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 12, p. 2576-2582Article in journal (Refereed)
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

  • 18.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Osterlund, Maria
    University Holding, Teknikringen 7, SE-581 83 Linköping, Sweden.
    Persson, Egon
    Vascular Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
    Freskgard, P.-O.
    Freskgård, P.-O., Protein Biotechnology, Novo Nordisk A/S, Novo Allé, DK-2880 Bagsværd, Denmark, Molecular Biology, Maxygen ApS, DK-2970, Hørsholm, 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.
    Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains2003In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1648, no 1-2, p. 12-16Article in journal (Refereed)
    Abstract [en]

    We have used the site-directed labeling approach to study the Ca 2+-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca2+ binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the ?-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca2+ titration is initiated upon Ca2+ binding to EGF1, the domain containing the site of highest Ca2+ affinity. Besides we showed that a Ca 2+-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca2+ binding to the PD seems to be of some importance for the docking of this domain to sTF. © 2003 Elsevier Science B.V. All rights reserved.

  • 19.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Egon
    Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 349, no 3, p. 1111-1116Article in journal (Refereed)
    Abstract [en]

    Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

  • 20.
    Carlsson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Persson, Egon
    Haemostasis Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
    Østergaard, Henrik
    Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
    Lindgren, Mikael
    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.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching2011In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, p. 545-549Article in journal (Refereed)
    Abstract [en]

    The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.

    Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

  • 21.
    Carlsson, Uno
    et al.
    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.
    Lindgren, M
    Persson, M
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D
    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.
    Aggregation is site-specific in carbonic anhydrase and is prevented by GroEL: The interaction leads to a more flexible structure of both the protein substrate and the chaperonin.2000In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 78, no 1, p. 202Pos-Conference paper (Other academic)
  • 22.
    Carlsson, Uno
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Magnusson, Thomas
    Linköping University, Department of Management and Engineering, Project management, Innovations and Entrepreneurship . Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Magdalena
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    To design a novel protein: A CDIO experience in Molecular Biotechnology at Linköping University2006Conference paper (Other academic)
  • 23. Frantz, S.E.A.
    et al.
    Mikael, L.A.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Quantum efficiency and two-photon absorption cross-section of conjugated polyelectrolytes used for protein conformation measurements with applications on amyloid structures2007In: Chemical Physics, ISSN 0301-0104, E-ISSN 1873-4421, Vol. 336, no 2-3, p. 121-126Article in journal (Refereed)
    Abstract [en]

    Amyloid diseases such as Alzheimer's and spongiform encephalopathies evolve from aggregation of proteins due to misfolding of the protein structure. Early disease handling require sophisticated but yet simple techniques to follow the complex properties of the aggregation process. Conjugated polyelectrolytes (CPEs) have shown promising capabilities acting as optical biological sensors, since they can specifically bind to polypeptides both in solution and in solid phase. The structural changes in biomolecules can be monitored by changes of the optical spectra of the CPEs, both in absorption and emission modes. Notably, the studied CPEs possess multi-photon excitation capability, making them potential for in vivo imaging using laser scanning microscopy. Aggregation of proteins depends on concentration, temperature and pH. The optical effect on the molecular probe in various environments must also be investigated if applied in these environments. Here we present the results of quantum efficiency and two-photon absorption cross-section of three CPEs: POMT, POWT and PTAA in three different pH buffer systems. The extinction coefficient and quantum efficiency were measured. POMT was found to have the highest quantum efficiency being approximately 0.10 at pH 2.0. The two-photon absorption cross-section was measured for POMT and POWT and was found to be more than 18-25 times and 7-11 times that of Fluorescein, respectively. We also show how POMT fluorescence can be used to distinguish conformational differences between amyloid fibrils formed from reduced and non-reduced insulin in spectrally resolved images recorded with a laser scanning microscope using both one- and two-photon excitation. © 2007 Elsevier B.V. All rights reserved.

  • 24.
    Fyrner, Timmy
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering.
    Magnusson, Karin
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Aili, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Sensor Science and Molecular Physics. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Derivatization of a bioorthogonal protected trisaccharide linker: towards multimodal tools for chemical biology2012In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 6, p. 1333-1340Article in journal (Refereed)
    Abstract [en]

    When cross-linking biomolecules to surfaces or to other biomolecules, the use of appropriate spacer molecules is of great importance. Mimicking the naturally occurring spacer molecules will give further insight into their role and function, possibly unveil important issues regarding the importance of the specificity of carbohydrate-based anchor moieties, in e.g., glycoproteins and glycosylphosphatidylinositols. Herein, we present the synthesis of a lactoside-based trisaccharide, potentially suitable as a heterobifunctional bioorthogonal linker molecule whereon valuable chemical handles have been conjugated. An amino-derivative having thiol functionality shows promise as novel SPR-surfaces. Furthermore, the trisaccharide has been conjugated to a cholesterol moiety in combination with a fluorophore which successfully assemble on the cell surface in lipid microdomains, possibly lipid-rafts. Finally, a CuI-catalyzed azide-alkyne cycloaddition reaction (CuAAC) confirms the potential use of oligosaccharides as bioorthogonal linkers in chemical biology.

  • 25.
    Gabrielsson, Erik O
    et al.
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    Tybrandt, Klas
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Berggren, Magnus
    Linköping University, Department of Science and Technology. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Spatially Controlled Amyloid Reactions Using Organic Electronics2010In: SMALL, ISSN 1613-6810, Vol. 6, no 19, p. 2153-2161Article in journal (Refereed)
    Abstract [en]

    Abnormal protein aggregates, so called amyloid fibrils, are mainly known as pathological hallmarks of a wide range of diseases, but in addition these robust well-ordered self-assembled natural nanostructures can also be utilized for creating distinct nanomaterials for bioelectronic devices. However, current methods for producing amyloid fibrils in vitro offer no spatial control. Herein, we demonstrate a new way to produce and spatially control the assembly of amyloid-like structures using an organic electronic ion pump (OEIP) to pump distinct cations to a reservoir containing a negatively charged polypeptide. The morphology and kinetics of the created proteinaceous nanomaterials depends on the ion and current used, which we leveraged to create layers incorporating different conjugated thiophene derivatives, one fluorescent (p-FTAA) and one conducting (PEDOT-S). We anticipate that this new application for the OEIP will be useful for both biological studies of amyloid assembly and fibrillogenesis as well as for creating new bioelectronic nanomaterials and devices.

  • 26.
    Groenning, Minna
    et al.
    University of Copenhagen.
    Campos, Raul I
    Linköping University, Department of Physics, Chemistry and Biology, Protein Science. Linköping University, Faculty of Science & Engineering.
    Fagerberg, Christina
    Vejle Hospital.
    Aamann Rasmussen, Anders
    Vejle Hospital.
    Eriksen, Ulrik H
    Vejle Hospital.
    Powers, Evan T
    Scripps Research Institute.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Thermodynamic stability and denaturation kinetics of a benign natural transthyretin mutant identified in a Danish kindred2011In: AMYLOID-JOURNAL OF PROTEIN FOLDING DISORDERS, ISSN 1350-6129, Vol. 18, no 2, p. 35-46Article in journal (Refereed)
    Abstract [en]

    The disease phenotype of transthyretin (TTR) is dramatically influenced by single point mutations in the TTR gene. Herein, we report on a novel mutation D99N (Asp99Asn) in TTR found in a Danish kindred. None of the family members carrying this mutation have so far shown any clinical signs of amyloidosis. One carrier found compound heterozygous for TTR D99N and L111M (Leu111Met) associated with cardiac amyloid is asymptomatic (42 years). Disease severity can often be linked to both the kinetics of fibril formation and the degree of destabilisation of the native state. In this study, we show that the thermodynamic stability and rate of tetramer dissociation of the variant TTR D99N is unchanged or slightly more stable than wild type (WT) TTR. Furthermore, the in vitro fibrillation kinetics of the variant reveals an unchanged or slightly suppressed tendency to form fibrils compared to WT. Thus, the in vitro experiments support the lack of clinical symptoms observed so far for the TTR D99N carriers. In line with this, studies on kinetic stability and fibrillation kinetics reveal indistinguishable stability of TTR heterotetramers D99N/L111M compared to the heterotetramers WT/L111M. In conclusion, TTR D99N is predicted to be a non-pathogenic benign mutation with WT properties.andlt;/.

  • 27.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Novel Conformational Probes for Protein Misfolding Diseases2005In: Keystone Symposia, Molecular Mechanisms of Transmissible Spongiform Encephalopathies,2005, 2005Conference paper (Other academic)
    Abstract [en]

    Invited Lecture  

  • 28.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Protein folding, misfolding and disease2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 16, p. 2579-2580Article in journal (Refereed)
  • 29.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    The bloody path of amyloids and prions2007In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 5, no 6, p. 1136-1138Other (Other academic)
    Abstract [en]

    [No abstract available]

  • 30.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    The dynamic amyloid landscape2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no Suppl. 1, p. 14-14Article in journal (Other academic)
    Abstract [en]

    n/a

  • 31.
    Hammarström, Per
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Ali, Malik M
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mishra, Rajesh
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Salagic, Belma
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Svensson, Samuel
    AstraZeneca RandD.
    Tengvall, Pentti
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils-Genesis of an Amyloid World?2011In: Origins of life and evolution of the biosphere, ISSN 0169-6149, E-ISSN 1573-0875, Vol. 41, no 4, p. 373-383Article in journal (Refereed)
    Abstract [en]

    Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65 degrees C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.

  • 32.
    Hammarström, Per
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Ali Malik, Muhammad
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Mishra, Rajesh
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Svensson, Samuel
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Tengvall, Pentti
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics . Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics . Linköping University, The Institute of Technology.
    A catalytic surface for amyloid fibril formation2008In: Journal of Physics, Conference Series, ISSN 1742-6588, E-ISSN 1742-6596, Vol. 100Article in journal (Refereed)
    Abstract [en]

    A hydrophobic surface incubated in a solution of protein molecules (insulin monomers) was made into a catalytic surface for amyloid fibril formation by repeatedly incubate, rinse and dry the surface. The present contribution describes how this unexpected transformation occurred and its relation to rapid fibrillation of insulin solutions in contact with the surface. A tentative model of the properties of the catalytic surface is given, corroborated by ellipsometric measurements of the thickness of the organic layer on the surface and by atomic force microscopy. The surfaces used were spontaneously oxidized silicon made hydrophobic through treatment in dichlorodimethylsilane.

  • 33.
    Hammarström, Per
    et al.
    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.
    Is the unfolded state the Rosetta Stone of the protein folding problem?2000In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 276, no 2, p. 393-398Article in journal (Refereed)
    Abstract [en]

    Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for ▀-sheet initiation and helical turns for a-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem. (C) 2000 Academic Press.

  • 34.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Jiang, X.
    Department of Chemistry, Skaggs Inst. for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road BCC265, San Diego, CA 92037, United States.
    Hurshman, A.R.
    Department of Chemistry, Skaggs Inst. for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road BCC265, San Diego, CA 92037, United States.
    Powers, E.T.
    Department of Chemistry, Skaggs Inst. for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road BCC265, San Diego, CA 92037, United States.
    Kelly, J.W.
    Department of Chemistry, Skaggs Inst. for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road BCC265, San Diego, CA 92037, United States.
    Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity2002In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, no SUPPL. 4, p. 16427-16432Conference paper (Other academic)
    Abstract [en]

    Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease, however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance-presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.

  • 35.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Protein denaturation and the denatured state2005In: Encyclopedia of Life Sciences, Wiley-Blackwell , 2005Chapter in book (Other (popular science, discussion, etc.))
    Abstract [en]

    Protein denaturation experiments are routinely used to determine protein stability and to elucidate structural and dynamic effects of mutations, cofactors and ligands. Denatured states of proteins have gained wide interest in recent years owing to their fundamental importance in a wide variety of phenomena such as deciphering the protein folding problem and the molecular understanding of many diseases.

  • 36.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Kalman, B.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Pyrene Excimer Fluorescence as a Proximity Probe for Investigation of Residual Structure in the Unfolded State of Human Carbonic Anhydrase II1997In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 420, p. 63-68Article in journal (Refereed)
  • 37.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Mårtensson, Lars-Göran
    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.
    Lindgren, M
    High-resolution probing of local conformational changes in proteins by the use of multiple labeling: Unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes2001In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 80, no 6, p. 2867-2885Article in journal (Refereed)
    Abstract [en]

    Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 Angstrom from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.

  • 38.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, M
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Compactness measurements at unfolding of carbonic anhydrase by Trp-AEDANS fluorescence energy transfer. Evidence for forced unfolding by GroEL.2000In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 78, no 1, p. 203Pos-Conference paper (Other academic)
  • 39.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Protein compactness measured by fluorescence resonance energy transfer - Human carbonic anhydrase II Is considerably expanded by the interaction of GroEL2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 24, p. 21765-21775Article in journal (Refereed)
    Abstract [en]

    Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nn (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label, Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL, It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased similar to2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to similar to 117,000 Angstrom (3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity, Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarstrom, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.

  • 40.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Freskgård, Per-Ola
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Andersson, D.
    Jonsson, Bengt-Harald
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology .
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Structural mapping of an aggregation nucleation site in a molten-globule intermediate1999In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 274, p. 32897-32903Article in journal (Refereed)
  • 41.
    Hammarström, Per
    et al.
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Persson, Malin
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Lindgren, M.
    Carlsson, Uno
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Protein substrate binding induces conformational changes in the chaperonin GroEL: A suggested mechanism for unfoldase activity2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 30, p. 22832-22838Article in journal (Refereed)
    Abstract [en]

    Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxy1-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.

  • 42.
    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)
  • 43.
    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.

  • 44.
    Hammarström, Per
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Simon, Rozalyn
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Åslund, Andreas
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
    A Fluorescent Pentameric Thiophene Derivative Detects in Vitro-Formed Prefibrillar Protein Aggregates2010In: BIOCHEMISTRY, ISSN 0006-2960, Vol. 49, no 32, p. 6838-6845Article in journal (Refereed)
    Abstract [en]

    Protein aggregation is associated with a wide range of diseases, and molecular probes that are able to detect a diversity of misfolded protein assemblies are of great importance. The identification of prefibrillar states preceding the formation of well-defined amyloid fibrils is of particular interest both because of their likely role in the mechanism of fibril formation and because of the growing awareness that these species are likely to play a critical role in the pathogenesis of protein deposition diseases. Herein, we explore the use of an anionic oligothiophene derivative, p-FTAA, for detection of prefibrillar protein aggregates during in vitro fibrillation of three different amyloidogenic proteins (insulin, lysozyme, and prion protein). p-FTAA generally detected prefibrillar protein aggregates that could not be detected by thioflavine T fluorescence and in addition showed high fluorescence when bound to mature fibrils. Second, the kinetics of protein aggregation or the formation of amyloid fibrils of insulin was not extensively influenced by the presence of various concentrations of p-FTAA. These results establish the use of p-FTAA as an additional tool for studying the process of protein aggregation.

  • 45.
    Herland, Anna
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics . Linköping University, The Institute of Technology.
    Björk, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics . Linköping University, The Institute of Technology.
    Nilsson, K. Peter R.
    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.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
    Electroactive luminescent self-assembled bio-organic nanowires: Integration of semiconducting oligoelectrolytes within amyloidogenic proteins2005In: Advanced Materials, ISSN 0935-9648, Vol. 17, no 12, p. 1466-1471Article in journal (Refereed)
  • 46.
    Herland, Anna
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics . Linköping University, The Institute of Technology.
    Nilsson, K. Peter R.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics . Linköping University, The Institute of Technology.
    Olsson, Johan D. M.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . 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.
    Synthesis of a regioregular zwitterionic conjugated oligoelectrolyte, usable as an optical probe for detection of amyloid fibril formation at acidic pH2005In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 7, p. 2317-2323Article in journal (Refereed)
    Abstract [en]

    Changes of the optical properties of conjugated polyelectrolytes have been utilized to monitor noncovalent interactions between biomolecules and the conjugated polyelectrolytes in sensor applications. A regioregular, zwitterionic conjugated oligoelectrolyte was synthesized in order to create a probe with a defined set of optical properties and hereby facilitate interpretation of biomolecule−oligoelectrolyte interactions. The synthesized oligoelectrolyte was used at acidic pH as a novel optical probe to detect amyloid fibril formation of bovine insulin and chicken lysozyme. Interaction of the probe with formed amyloid fibrils results in changes of the geometry and the electronic structure of the oligoelectrolyte chains, which were monitored with absorption and emission spectroscopy.

  • 47. Huber, M.
    et al.
    Lindgren, M.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mårtensson, Lars-Göran
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Eaton, G.R.
    Eaton, S.S.
    Phase memory relaxation times of spin labels in human carbonic anhydrase II: Pulsed EPR to determine spin label location2001In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 94, no 3, p. 245-256Article in journal (Refereed)
    Abstract [en]

    Phase memory relaxation times (TM or T2) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters TM and x. TM values of seven positions are between 1.6 ╡s for the most buried residue (L79C) and 4.7 ╡s for a residue at the protein surface (W245C). In deuteriated buffer, longer TM are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose TM as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism. ⌐ 2001 Elsevier Science B.V. All rights reserved.

  • 48.
    Huber, M
    et al.
    Leiden Univ, Dept Mol Phys, NL-2300 RA Leiden, Netherlands Inst Phys & Measurement Technol, Linkoping, Sweden.
    Owenius, Rikard
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology.
    Hammarström, Per
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Mårtensson, Lars-Göran
    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.
    High-field (95 GHz) EPR on spin labels in human carbonic anhydrase II: Perspectives of pulsed and CW EPR2000In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 78, no 1, p. 182Pos-Conference paper (Other academic)
  • 49.
    Höst, Gunnar
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Mårtensson, Lars-Göran
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. 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.
    Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains2006In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, ISSN 1570-9639, Vol. 1764, no 10, p. 1601-1606Article in journal (Refereed)
    Abstract [en]

    The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

  • 50.
    Jinnelöv, Anders
    Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
    Investigation of small molecules binding to UDP-galactose 4'-epimerase: A validated drug target for Trypanosoma brucei, the parasite responsible for African Sleeping Sickness.2009Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs.

    TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness.

    The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.

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