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  • 1.
    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.

  • 2.
    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.

  • 3.
    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.

  • 4.
    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.

  • 5. 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.

  • 6.
    Arja, Katriann
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology. Linköping, .
    Sjölander, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Protein Science. Linköping University, Faculty of Science & Engineering. Linköping, .
    Åslund, Alma
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering. Linköping, .
    Prokop, Stefan
    Charite, Germany .
    Heppner, Frank L.
    Charite, Germany .
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering. Linköping, .
    Lindgren, Mikael
    Norwegian University of Science and Technology, Norway .
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Protein Science. Linköping University, Faculty of Science & Engineering. Linköping, .
    Åslund, Andreas
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering. Linköping, .
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, Faculty of Science & Engineering. Linköping, .
    Enhanced Fluorescent Assignment of Protein Aggregates by an Oligothiophene-Porphyrin-Based Amyloid Ligand2013In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 34, no 9, p. 723-730Article in journal (Refereed)
    Abstract [en]

    Fluorescent probes identifying protein aggregates are of great interest, as deposition of aggregated proteins is associated with many devastating diseases. Here, we report that a fluorescent amyloid ligand composed of two distinct molecular moieties, an amyloidophilic pentameric oligothiophene and a porphyrin, can be utilized for spectral and lifetime imaging assessment of recombinant A 1-42 amyloid fibrils and A deposits in brain tissue sections from a transgenic mouse model with Alzheimers disease pathology. The enhanced spectral range and distinct lifetime diversity of this novel oligothiopheneporphyrin-based ligand allow a more precise assessment of heterogeneous amyloid morphology compared with the corresponding oligothiophene dye.

  • 7.
    Babu Moparthi, Satish
    et al.
    Aix Marseille University, France.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vincentelli, Renaud
    University of Aix Marseille, France.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wenger, Jerome
    Aix Marseille University, France.
    Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, no 28386Article in journal (Refereed)
    Abstract [en]

    Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

  • 8.
    Babu Moparthi, Satish
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Institut Fresnel, CNRS UMR 7249, Aix-Marseille Université, Marseille, France.
    Sjölander, Daniel
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Villebeck, Laila
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Carlsson, Uno
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL2014In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 7, no 1, p. 1-15Article, review/survey (Refereed)
    Abstract [en]

    The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

  • 9.
    Bagheri, Maryam
    et al.
    Ilam University of Medical Science, Iran .
    Rezakhani, Arjang
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Turkina, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Roghani, Mehrdad
    Shahed University, Iran .
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mohseni, Simin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Amyloid Beta1-40-Induced Astrogliosis and the Effect of Genistein Treatment in Rat: A Three-Dimensional Confocal Morphometric and Proteomic Study2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10Article in journal (Refereed)
    Abstract [en]

    Astrocytes are highly involved in regulation and homeostasis of the extracellular environment in the healthy brain. In pathological conditions, these cells play a major role in the inflammatory response seen in CNS tissues, which is called reactive astrogliosis and includes hypertrophy and proliferation of astrocytes. Here, we performed 3D confocal microscopy to evaluate the morphological response of reactive astrocytes positive for glial fibrillary acidic protein (GFAP) in rats, to the presence of Aβ1–40 in the rat brain before and after treatment with genistein. In 50 astrocytes per animal, we measured the volume and surface area for the nucleus, cell body, the entire cell, the tissue covered by single astrocytes and quantified the number and length of branches, the density of the astrocytes and the intensity of GFAP immunoreactivity. Injecting Aβ1–40 into the brain of rats caused astrogliosis indicated by increased values for all measured parameters. Mass spectrometric analysis of hippocampal tissue in Aβ1–40-injected brain showed decreased amounts of tubulins, enolases and myelin basic protein, and increased amounts of dihydropyrimidinase-related protein 2. In Aβ1–40-injected rats pretreated with genistein, GFAP intensity was decreased to the sham-operated group level, and Aβ1–40-induced astrogliosis was significantly ameliorated.

  • 10.
    Bednarska, Natalia G.
    et al.
    KULeuven, Belgium; VIB, Belgium.
    van Eldere, Johan
    KULeuven, Belgium.
    Gallardo, Rodrigo
    VIB, Belgium; KULeuven, Belgium.
    Ganesan, Ashok
    VIB, Belgium; KULeuven, Belgium.
    Ramakers, Meine
    VIB, Belgium; KULeuven, Belgium.
    Vogel, Isabel
    KULeuven, Belgium.
    Baatsen, Pieter
    VIB11, Belgium; KULeuven, Belgium.
    Staes, An
    VIB, Belgium; University of Ghent, Belgium.
    Goethals, Marc
    VIB, Belgium; University of Ghent, Belgium.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, K. Peter R.
    VIB, Belgium.
    Gevaert, Kris
    VIB, Belgium; University of Ghent, Belgium.
    Schymkowitz, Joost
    VIB, Belgium; KULeuven, Belgium.
    Rousseau, Frederic
    VIB, Belgium; KULeuven, Belgium.
    Protein aggregation as an antibiotic design strategy2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 99, no 5, p. 849-865Article in journal (Refereed)
    Abstract [en]

    Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

  • 11.
    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.

  • 12.
    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.

  • 13.
    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.

  • 14.
    Campos Melo, Raúl Ivan
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wu, Xiongyu
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Elgland, Mathias
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Novel Trans-Stilbene-based Fluorophores as Probes for Spectral Discrimination of Native and Protofibrillar Transthyretin2016In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 7, no 7, p. 924-940Article in journal (Refereed)
    Abstract [en]

    Accumulation of misfolded transthyretin (TTR) as amyloid fibrils causes various human disorders. Native transthyretin is a neurotrophic protein and is a putative extracellular molecular chaperone. Several fluorophores have been shown in vitro to bind selectively to native TTR. Other compounds, such as thioflavin T, bind TTR amyloid fibrils. The probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to both native and fibrillar TTR, becoming highly fluorescent, but with indistinguishable emission spectra for native and fibrillar TTR. Herein we report our efforts to develop a fluorescent small molecule capable of binding both native and misfolded protofibrillar TTR, providing distinguishable emission spectra. We used microwave synthesis for efficient production of a small library of trans-stilbenes and fluorescence spectral screening of their binding properties. We synthesized and tested 22 trans-stilbenes displaying a variety of functional groups. We successfully developed two naphthyl-based trans-stilbenes probes that detect both TTR states at physiological concentrations. The compounds bound with nanomolar to micromolar affinities and displayed distinct emission maxima upon binding native or misfolded protofibrillar TTR (>100 nm difference). The probes were mainly responsive to environment polarity providing evidence for the divergent hydrophobic structure of the binding sites of these protein conformational states. Furthermore, we were able to successfully use one of these probes to quantify the relative amounts of native and protofibrillar TTR in a dynamic equilibrium. In conclusion, we identified two trans-stilbene-based fluorescent probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (11) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (14), that bind native and protofibrillar TTR, providing a wide difference in emission maxima allowing conformational discrimination by fluorescence spectroscopy. We expect these novel molecules to serve as important chemical biology research tools in studies of TTR folding and misfolding.

  • 15.
    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)
  • 16.
    Ceasar (Berg), Ina
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Protein Science. Linköping University, The Institute of Technology.
    Jonsson, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Protein Science. 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, Protein Science. Linköping University, The Institute of Technology.
    Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2Article in journal (Refereed)
    Abstract [en]

    The pathology of Alzheimers disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-beta (A beta) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic A beta amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate A beta toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of A beta deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to A beta deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of A beta(1-42) in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant A beta(1-42). Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of A beta, resulting in a reduced neurotoxicity in Drosophila.

  • 17. 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.

  • 18.
    Fritschi, Sarah K.
    et al.
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany; University of Tubingen, Germany.
    Cintron, Amarallys
    Emory University, GA 30329 USA.
    Ye, Lan
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany; University of Tubingen, Germany.
    Mahler, Jasmin
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany; University of Tubingen, Germany.
    Buehler, Anika
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany.
    Baumann, Frank
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany.
    Neumann, Manuela
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Walker, Lary C.
    Emory University, GA 30329 USA; Emory University, GA 30322 USA.
    Jucker, Mathias
    German Centre Neurodegenerat Disease DZNE, Germany; University of Tubingen, Germany .
    A beta seeds resist inactivation by formaldehyde2014In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 128, no 4, p. 477-484Article in journal (Refereed)
    Abstract [en]

    Cerebral beta-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated beta-amyloid (A beta) into young, pre-depositing A beta precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated A beta itself. Here we report that the beta-amyloid-inducing activity of Alzheimers disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated A beta are maintained in fixed tissues. The resistance of A beta seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of A beta aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

  • 19.
    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.

  • 20.
    Fändrich, M.
    et al.
    Ulm Univ, Germany.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Bockmann, A.
    Univ Lyon, France.
    LeVine, H. III
    Univ Kentucky, KY 40536 USA; Univ Kentucky, KY USA.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Amyloid fibril polymorphism: a challenge for molecular imaging and therapy2018In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 283, no 3, p. 218-237Article in journal (Refereed)
    Abstract [en]

    The accumulation of misfolded proteins (MPs), both unique and common, for different diseases is central for many chronic degenerative diseases. In certain patients, MP accumulation is systemic (e.g. TTR amyloid), and in others, this is localized to a specific cell type (e.g. Alzheimers disease). In neurodegenerative diseases, NDs, it is noticeable that the accumulation of MP progressively spreads throughout the nervous system. Our main hypothesis of this article is that MPs are not only markers but also active carriers of pathogenicity. Here, we discuss studies from comprehensive molecular approaches aimed at understanding MP conformational variations (polymorphism) and their bearing on spreading of MPs, MP toxicity, as well as MP targeting in imaging and therapy. Neurodegenerative disease (ND) represents a major and growing societal challenge, with millions of people worldwide suffering from Alzheimers or Parkinsons diseases alone. For all NDs, current treatment is palliative without addressing the primary cause and is not curative. Over recent years, particularly the shape-shifting properties of misfolded proteins and their spreading pathways have been intensively researched. The difficulty in addressing ND has prompted most major pharma companies to severely downsize their nervous system disorder research. Increased academic research is pivotal for filling this void and to translate basic research into tools for medical professionals. Recent discoveries of targeting drug design against MPs and improved model systems to study structure, pathology spreading and toxicity strongly encourage future studies along these lines to provide an opportunity for selective imaging, prognostic diagnosis and therapy.

  • 21.
    Gabrielsson, Erik
    et al.
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, Faculty of Science & Engineering.
    Armgarth, Astrid
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Berggren, Magnus
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, Faculty of Science & Engineering.
    Spatiotemporal Control of Amyloid-Like A Plaque Formation Using a Multichannel Organic Electronic Device2016In: Macromolecular materials and engineering (Print), ISSN 1438-7492, E-ISSN 1439-2054, Vol. 301, no 4, p. 359-363Article in journal (Refereed)
    Abstract [en]

    We herein report on an iontronic device to drive and control A1-40 and A1-42 fibril formation. This system allows kinetic control of A aggregation by regulation of H+ flows. The formed aggregates show both nanometer-sized fibril structure and microscopic growth, thus mimicking senile plaques, at the H+-outlet. Mechanistically we observed initial accumulation of A1-40 likely driven by electrophoretic migration which preceded nucleation of amyloid structures in the accumulated peptide cluster.

  • 22.
    Gabrielsson, Erik O.
    et al.
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, The Institute of Technology.
    Armgarth, Astrid
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Nilsson, K. Peter N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Berggren, Magnus
    Linköping University, Department of Science and Technology, Physics and Electronics. Linköping University, The Institute of Technology.
    Controlled Microscopic Formation of Amyloid-Like Aβ Aggregates Using an Organic Electronic DeviceManuscript (preprint) (Other academic)
    Abstract [en]

    Alzheimer’s disease (AD), primarily associated with formation of fibrillar amyloid-beta peptide (Aβ) aggregates in the brain, is one of the most common old-age diseases. It is therefore crucial with an elevated scientific interest in Aβ, and its fundamental properties in a wide sense, to develop efficient methods for early detection and to combat AD. For the development of new techniques, both for AD detection and prevention, researchers are dependent on either tissue samples from deceased patients, animal models or in vitro systems. In vitro systems, such as producing protein aggregates of the Aβ-peptide in a test tube by incubation under denaturing conditions, offers us a simple but rather blunt tool for evaluating aggregation inhibition caused by compounds or to investigate new detection methods. We recently introduced the organic electronic ion pump (OEIP) as a method for creating amyloid-like aggregates at high spatiotemporal control as compared to the resulting aggregates manufactured using regular test tube-conditions. Combined with a fluorescent probe that is specific for the fibrillar aggregated form of misfolded peptides commonly seen in AD, this allowed us to control and to monitor the aggregation of a model peptide system in a highly confined space.

    To further elaborate the functionality of the OEIP together with amyloid-specific probes, we here present experiments demonstrating electronically controlled micron sized formation of Aβ-aggregates with morphologies ranging from fine fibers, to bundles of fibers, and thick mesh-like fiber structures. We foresee that the methodology can be implemented in multi array systems that can be utilized for studies of protein aggregation in confined spaces or together with cultured cells, as well as for the development of screening platforms for assessment of molecules influencing the Aβ-aggregation process.

  • 23.
    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.

  • 24.
    Gallardo, Rodrigo
    et al.
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Ramakers, Meine
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    De Smet, Frederik
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Claes, Filip
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Khodaparast, Ladan
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Khodaparast, Laleh
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Couceiro, Jose R.
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Langenberg, Tobias
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Siemons, Maxime
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Young, Laurence J.
    University of Cambridge, England; University of Leeds, England; University of Leeds, England.
    Laine, Romain F.
    University of Cambridge, England.
    Young, Lydia
    University of Cambridge, England; University of Leeds, England; University of Leeds, England.
    Radaelli, Enrico
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Benilova, Iryna
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Kumar, Manoj
    Katholieke University of Leuven, Belgium.
    Staes, An
    VIB, Belgium; University of Ghent, Belgium.
    Desager, Matyas
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Beerens, Manu
    Katholieke University of Leuven, Belgium.
    Vandervoort, Petra
    Katholieke University of Leuven, Belgium.
    Luttun, Aernout
    Katholieke University of Leuven, Belgium.
    Gevaert, Kris
    VIB, Belgium; University of Ghent, Belgium.
    Bormans, Guy
    Katholieke University of Leuven, Belgium.
    Dewerchin, Mieke
    Katholieke University of Leuven, Belgium; VIB, Belgium.
    Van Eldere, Johan
    Katholieke University of Leuven, Belgium.
    Carmeliet, Peter
    Katholieke University of Leuven, Belgium; VIB, Belgium.
    Vande Velde, Greetje
    Katholieke University of Leuven, Belgium.
    Verfaillie, Catherine
    Katholieke University of Leuven, Belgium.
    Kaminski, Clemens F.
    University of Cambridge, England.
    De Strooper, Bart
    VIB Centre Biol Disease, Belgium; Katholieke University of Leuven, Belgium; Katholieke University of Leuven, Belgium.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Serpell, Louise
    University of Sussex, England.
    Schymkowitz, Joost
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    Rousseau, Frederic
    VIB Switch Lab, Belgium; Katholieke University of Leuven, Belgium.
    De novo design of a biologically active amyloid2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 354, no 6313, p. 720-+Article in journal (Refereed)
    Abstract [en]

    Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional-determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

  • 25.
    Groenning, Minna
    et al.
    University of Copenhagen, Denmark.
    Campos Melo, Raul Ivan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hirschberg, Daniel
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vestergaard, Bente
    University of Copenhagen, Denmark; University of Copenhagen, Denmark.
    Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, no 11443Article in journal (Refereed)
    Abstract [en]

    Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

  • 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.
    Hahn, Katharina
    et al.
    Christian Albrechts University of Kiel, Germany.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Urban, Peter
    Institute Pathol and Dermatopathol, Germany.
    Ruediger Meliss, Rolf
    Institute Dermatopathol, Germany.
    Behrens, Hans-Michael
    Christian Albrechts University of Kiel, Germany.
    Krueger, Sandra
    Christian Albrechts University of Kiel, Germany.
    Roecken, Christoph
    Christian Albrechts University of Kiel, Germany.
    Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome2017In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 24, no 2, p. 78-86Article in journal (Refereed)
    Abstract [en]

    Transthyretin-derived (ATTR) amyloidosis is a frequent finding in carpal tunnel syndrome. We tested the following hypotheses: the novel fluorescent amyloid ligand heptameric formic thiophene acetic acid (h-FTAA) has a superior sensitivity for the detection of amyloid compared with Congo red-staining; Amyloid load correlates with patient gender and/or patient age. We retrieved 208 resection specimens obtained from 184 patients with ATTR amyloid in the carpal tunnel. Serial sections were stained with Congo red, h-FTAA and an antibody directed against transthyretin (TTR). Stained sections were digitalized and forwarded to computational analyses. The amount of amyloid was correlated with patient demographics. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Congo red-staining combined with fluorescence microscopy was significantly less sensitive than h-FTAA-fluorescence and TTR-immunostaining: the highest percentage area was found in TTR-immunostained sections, followed by h-FTAA and Congo red. The Pearson correlation coefficient was .8 (Congo red vs. h-FTAA) and .9 (TTR vs. h-FTAA). Amyloid load correlated with patient gender, anatomical site and patient age. h-FTAA is a highly sensitive method to detect even small amounts of ATTR amyloid in the carpal tunnel. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

  • 28.
    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  

  • 29.
    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)
  • 30.
    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]

  • 31.
    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

  • 32.
    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.

  • 33.
    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.

  • 34.
    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.

  • 35.
    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.

  • 36.
    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.

  • 37.
    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)
  • 38.
    Hammarström, Per
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Porcine prion protein amyloid2015In: Prion, ISSN 1933-6896, E-ISSN 1933-690X, Vol. 9, no 4, p. 266-277Article in journal (Refereed)
    Abstract [en]

    Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

  • 39.
    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.

  • 40.
    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)
  • 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.
    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.

  • 42.
    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)
  • 43.
    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.

  • 44.
    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)
  • 45.
    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.

  • 46.
    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.

  • 47.
    Heilbronner, Goetz
    et al.
    University of Tubingen, Germany .
    Eisele, Yvonne S.
    University of Tubingen, Germany .
    Langer, Franziska
    University of Tubingen, Germany .
    Kaeser, Stephan A.
    University of Tubingen, Germany .
    Novotny, Renata
    University of Tubingen, Germany .
    Nagarathinam, Amudha
    University of Tubingen, Germany .
    Åslund, Andreas
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Jucker, Mathias
    University of Tubingen, Germany .
    Seeded strain-like transmission of beta-amyloid morphotypes in APP transgenic mice2013In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 14, no 11, p. 1017-1022Article in journal (Refereed)
    Abstract [en]

    The polymorphic beta-amyloid lesions present in individuals with Alzheimers disease are collectively known as cerebral beta-amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop beta-amyloid depositions that differ in morphology, binding of amyloid conformation-sensitive dyes, and A beta 40/A beta 42 peptide ratio. To determine the nature of such beta-amyloid morphotypes, beta-amyloid-containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced beta-amyloid deposition with the morphological, conformational, and A beta 40/A beta 42 ratio characteristics of beta-amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced b-amyloid deposits with the characteristics of beta-amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced beta-amyloid deposits, although less prominent, and the induced deposits were similar to the beta-amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated A beta in APP transgenic mice can be maintained by seeded conversion.

  • 48.
    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)
  • 49.
    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.

  • 50.
    Herrmann, Uli S.
    et al.
    University of Zurich Hospital, Switzerland.
    Schuetz, Anne K.
    ETH, Switzerland.
    Shirani, Hamid
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Huang, Danzhi
    University of Zurich, Switzerland.
    Saban, Dino
    University of Zurich Hospital, Switzerland.
    Nuvolone, Mario
    University of Zurich Hospital, Switzerland.
    Li, Bei
    University of Zurich Hospital, Switzerland.
    Ballmer, Boris
    University of Zurich Hospital, Switzerland.
    Åslund, Andreas
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Mason, Jeffrey
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Rushing, Elisabeth
    University of Zurich Hospital, Switzerland.
    Budka, Herbert
    University of Zurich Hospital, Switzerland.
    Nyström, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hammarström, Per
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Boeckmann, Anja
    University of Lyon 1, France.
    Caflisch, Amedeo
    University of Zurich, Switzerland.
    Meier, Beat H.
    ETH, Switzerland.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Hornemann, Simone
    University of Zurich Hospital, Switzerland.
    Aguzzi, Adriano
    University of Zurich Hospital, Switzerland.
    Structure-based drug design identifies polythiophenes as antiprion compounds2015In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 7, no 299, p. 299ra123-Article in journal (Refereed)
    Abstract [en]

    Prions cause transmissible spongiform encephalopathies for which no treatment exists. Prions consist of PrPSc, a misfolded and aggregated form of the cellular prion protein (PrPC). We explore the antiprion properties of luminescent conjugated polythiophenes (LCPs) that bind and stabilize ordered protein aggregates. By administering a library of structurally diverse LCPs to the brains of prion-infected mice via osmotic minipumps, we found that antiprion activity required a minimum of five thiophene rings bearing regularly spaced carboxyl side groups. Solid-state nuclear magnetic resonance analyses and molecular dynamics simulations revealed that anionic side chains interacted with complementary, regularly spaced cationic amyloid residues of model prions. These findings allowed us to extract structural rules governing the interaction between LCPs and protein aggregates, which we then used to design a new set of LCPs with optimized binding. The new set of LCPs showed robust prophylactic and therapeutic potency in prion-infected mice, with the lead compound extending survival by greater than80% and showing activity against both mouse and hamster prions as well as efficacy upon intraperitoneal administration into mice. These results demonstrate the feasibility of targeted chemical design of compounds that may be useful for treating diseases of aberrant protein aggregation such as prion disease.

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