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  • 1.
    Anandapadamanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Kyriakidis, Nikolaos C.
    Karolinska Univ Hosp, Sweden; UDLA, Ecuador.
    Csizmok, Veronika
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallenhammar, Amélie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Espinosa, Alexander C.
    Karolinska Univ Hosp, Sweden.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Round, Adam R.
    Grenoble Outstn, France; European XFEL GmbH, Germany.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Univ Sydney, Australia.
    Moche, Martin
    Karolinska Inst, Sweden.
    Wahren-Herlenius, Marie
    Karolinska Univ Hosp, Sweden.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E22019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 294, no 30, p. 11404-11419Article in journal (Refereed)
    Abstract [en]

    The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-angstrom crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called linchpin residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.

  • 2.
    Anandapadmanaban, Madhanagopal
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Pilstål, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Trewhella, Jill
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. University of Sydney, Australia.
    Moche, Martin
    Karolinska Institute, Sweden.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, p. 1311-1321Article in journal (Refereed)
    Abstract [en]

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

  • 3.
    Andrésen, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Niklasson, Markus
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Cassman Eklöf, Sofie
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 32017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 7, article id e0181721Article in journal (Refereed)
    Abstract [en]

    Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond time-scale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.

  • 4.
    Blissing, Annica
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Thiopurine S-methyltransferase - characterization of variants and ligand binding2017Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects.

    Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function.

    Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (KD ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

  • 5.
    Conti, Luca
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Renhorn, Jakob
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Gabrielsson, Anders
    KTH Royal Institute Technology, Sweden.
    Turesson, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Liin, Sara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Lindahl, Erik
    KTH Royal Institute Technology, Sweden; Stockholm University, Sweden.
    Elinder, Fredrik
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 27562Article in journal (Refereed)
    Abstract [en]

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  • 6.
    Gustafsson, Robert
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology.
    Biophysical characterization of the *5 protein variant of human thiopurine methyltransferase by NMR spectroscopy2012Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site.

    Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability.

    Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.

  • 7.
    Howe, Christoph
    et al.
    Uppsala Univ, Sweden.
    Moparthi, Vamsi
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Uppsala Univ, Sweden.
    Ho, Felix M.
    Uppsala Univ, Sweden.
    Persson, Karina
    Umea Univ, Sweden.
    Stensjo, Karin
    Uppsala Univ, Sweden.
    The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria2019In: PLoS ONE, E-ISSN 1932-6203, Vol. 14, no 8, article id e0218300Article in journal (Refereed)
    Abstract [en]

    Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron-and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O-2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O-2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

  • 8.
    Javaherian, Anoosh D.
    et al.
    Division of Molecular Medicine, Department of Anesthesiolog, David Geffen School of Medicine, UCLA, Los Angeles, USA.
    Yusifov, Taleh
    Division of Molecular Medicine, Department of Anesthesiolog, David Geffen School of Medicine, UCLA, Los Angeles, USA.
    Pantazis, Antonios
    Division of Molecular Medicine, Department of Anesthesiolog, David Geffen School of Medicine, UCLA, Los Angeles, USA.
    Franklin, Sarah
    Division of Molecular Medicine, Department of Anesthesiolog, David Geffen School of Medicine, UCLA, Los Angeles, USA.
    Gandhi, Chris S.
    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.
    Olcese, Riccardo
    ivision of Molecular Medicine, Department of Anesthesiology, Cardiovascular Research Laboratory, and Brain Research Institute, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.
    Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 23, p. 20701-20709Article in journal (Refereed)
    Abstract [en]

    Large-conductance voltage- and Ca2+-dependent K+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca2+ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca2+ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca2+- and Mg2+-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba2+-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca2+-binding site (the “calcium bowl”) reduced the Ca2+ and abolished the Mg2+ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca2+-binding site and that Mg2+ can bind to the calcium bowl with less affinity than Ca2+. Dynamic light scattering analysis revealed a reversible Ca2+-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

  • 9.
    Karlsson, K Fredrik
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Dupertuis, M. A.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Physics of Nanostructures, CH‐1015 Lausanne, Switzerland.
    Oberli, D. Y.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Physics of Nanostructures, CH‐1015 Lausanne, Switzerland.
    Pelucchi, E.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Physics of Nanostructures, CH‐1015 Lausanne, Switzerland.
    Rudra, A.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Physics of Nanostructures, CH‐1015 Lausanne, Switzerland.
    Holtz, Per-Olof
    Linköping University, Department of Physics, Chemistry and Biology, Semiconductor Materials. Linköping University, Faculty of Science & Engineering.
    Kapon, E.
    Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Physics of Nanostructures, CH‐1015 Lausanne, Switzerland.
    Symmetry Elevation and Symmetry Breaking: Keys to Describe and Explain Excitonic Complexes in Semiconductor Quantum Dots2011Conference paper (Refereed)
    Abstract [en]

    The results of a group theoretical analysis of the excitonic fine structure are presented and compared with spectroscopic data on single quantum dots. The spectral features reveal the signatures of a symmetry higher than the crystal symmetry (C 3v ).  A consistent picture of the fine structure patterns for various exciton complexes is obtained with group theory and the concepts of symmetry elevation and symmetry breaking.

  • 10.
    Kontush, Anatol
    et al.
    National Institute for Health and Medical Research (INSERM), UMR-ICAN 1166, Paris, France // University of Pierre and Marie Curie - Paris 6, Paris, France // Pitié – Salpétrière University Hospital, Paris, France // ICAN, Paris, France.
    Lindahl, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
    Lhomme, Marie
    National Institute for Health and Medical Research (INSERM), UMR-ICAN 1166, Paris, France // University of Pierre and Marie Curie - Paris 6, Paris, France // Pitié – Salpétrière University Hospital, Paris, France // ICAN, Paris, France.
    Calabresi, Laura
    Department of Pharmacological and Biomolecular Sciences, Center E. Grossi Paoletti, University of Milan, Milan, Italy .
    Chapman, M John
    National Institute for Health and Medical Research (INSERM), UMR-ICAN 1166, Paris, France // University of Pierre and Marie Curie - Paris 6, Paris, France // Pitié – Salpétrière University Hospital, Paris, France // ICAN, Paris, France.
    Davidson, W Sean
    Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, 45237, USA.
    Structure of HDL: particle subclasses and molecular components2015In: High Density Lipoproteins – from biological understanding to clinical exploitation, Springer, 2015, Vol. 224, p. 3-51Chapter in book (Refereed)
    Abstract [en]

    A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.

  • 11.
    Lindberg, Max
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry.
    Fluorescent fusion proteins as probes to characterize tau fibril polymorphism2019Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Alzheimer's disease (AD) is a large and growing problem and while we today lack a full understanding of this disease, we know that the protein tau and the amyloid fibrils it forms play a central role in its development. We also know that these fibrils can have different morphologies in different diseases and that fibrils produced in vitro not necessarily adopt any of the morphologies found in patients. This means there is a need for more pathologically relevant fibrils in vitro to be able to understand this disease better. One approach to satisfy this need is to use fibrils found in patients as seeds and thus transfer their morphology to recombinantly purified protein. To facilitate this process this study has attempted to develop a way to differentiate between different fibril morphologies using a FRET based system. This involves fluorescent fusion proteins (tau-EXFPs) and fluorescent amyloid probes as well as seeding experiments with pseudo wild type tau (PWT) and tau with the P301L mutation. Greater differences in terms of fibrillation rates and ThT fluorescence between PWT and P301L was shown than previously reported between WT and P301L. They were also shown to differ in fibril morphology in TEM. The ThT fluorescence intensity was to a certain degree transferable from PWT to P301L by seeding. Furthermore, this study confirms that the tau-EXFP fusion protein can be incorporated into amyloid fibrils and strongly suggests that a FRET effect between EXFP and BTD14 (as well as X34 and ThT) can be achieved. It also demonstrates differences in FRET efficiency between PWT and P301L fibrils using FLIM. These results indicate that a FRET based approach could be a useful method to discern different fibril morphologies from each other, but further measurements and optimization are needed before this method could be reliably applied. The fusion proteins could also be used to investigate tau spreading in vivo, e.g. in D. melanogaster. To find suitable FRET partners to the fusion proteins, a ligand screen was conducted. This could be used as an alternative to the FRET method. With the right selection of fluorescent amyloid probes, a unique fingerprint for each fibril morphology could maybe be generated and fulfill the same intended function as the FRET method.

  • 12.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Quantitative analysis of conformational exchange contributions to H-1-N-15 multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, ISSN 0002-7863, Vol. 126, no 3, p. 928-935Article in journal (Refereed)
    Abstract [en]

    Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential H-1-N-15 multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the H-1 and N-15 chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential H-1-N-15 multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from N-15 off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the H-1 and N-15 spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the H-1 chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on H-1 single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.

  • 13.
    Lundström, Patrik
    et al.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Hansen, D. Flemming
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Kay, Lewis E.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively C-13 labeled samples2008In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 42, no 1, p. 35-47Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated C-13 spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (CO)-C-13 experiments however the large separation between (CO)-C-13 and C-13(alpha) chemical shifts offers hope that robust (CO)-C-13 dispersion profiles can be recorded on uniformly C-13 labeled samples, leading to the extraction of accurate (CO)-C-13 chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-C-13]-pyruvate and (NaHCO3,)-C-13 or uniformly labeled, generated from C-13-glucose. Very similar (CO)-C-13 chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

  • 14.
    Lundström, Patrik
    et al.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Hansen, D. Flemming
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Vallurupalli, Parmodh
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Kay, Lewis E.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 5, p. 1915-1926Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy can provide detailed information about low populated, invisible states of protein molecules, including backbone chemical shifts of the invisible conformer and bond vector orientations that can be used as structural constraints. Notably, the measurement of H-1(alpha) chemical shifts in excited protein states has not been possible to date because, in the absence of suitable labeling, the homonuclear proton scalar coupling network in side chains of proteins leads to a significant degradation in the performance of proton-based relaxation dispersion experiments. Here we have overcome this problem through a labeling scheme in which proteins are prepared with U-H-2 glucose and 50% D2O/50% H2O that results in cleuteration levels of between 50-88% at the C-beta carbon. Effects from residual H-1(alpha)-H-1(beta) scalar couplings can be suppressed through a new NMR experiment that is presented here. The utility of the methodology is demonstrated on a ligand binding exchanging system and it is shown that H-1(alpha) chemical shifts extracted from dispersion profiles are, on average, accurate to 0.03 ppm, an order of magnitude better than they can be predicted from structure using a database approach. The ability to measure H-1(alpha) chemical shifts of invisible conformers is particularly important because such shifts are sensitive to both secondary and tertiary structure. Thus, the methodology presented is a valuable addition to a growing list of experiments for characterizing excited protein states that are difficult to study using the traditional techniques of structural biology.

  • 15.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Mulder, Frans A. A.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 47, p. 16984-16989Article in journal (Refereed)
    Abstract [en]

    Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used C-13(alpha)(i - 1)/C-13(alpha)(i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc.), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of approximate to 20 mu s. The present results reveal that an entire alpha-helix undergoes partial unraveling in a transient and cooperative manner.

  • 16.
    Magnusson, Karin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Appelqvist, Hanna
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Cieslar-Pobuda, Artur
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Institute of Automatic Control, Silesian University of of TechnologyGliwice, Poland.
    Wigenius, Jens
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology. Carl Zeiss AB, Sweden.
    Karlsson, Thommie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Application Specialist Confocal Microscopy at Leica MicrosystemsIL, United States.
    Los, Marek Jan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Department of Pathology, Pomeranian Medical UniversitySzczecin, Poland.
    Kågedal, Bertil
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Jonasson, Jon
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Nilsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte2015In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 87, no 3, p. 262-272Article in journal (Refereed)
    Abstract [en]

    Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.

  • 17.
    Rydberg, David
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Dependence on pH of Structural and Dynamical Changes of a Calmodulin Domain Mutant2015Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis
    Abstract [en]

    Calmodulin (CaM) is a highly conserved protein able to bind Ca2+. When Ca2+ is bound the protein can bind and activate further proteins with several individual functions. CaM switches to a more open conformation when Ca2+-bound and is able to do so at a high rate. Little is known about the conformational switches between apo and Ca2+-bound states. A hypothesis suggests that protonation/deprotonation of a histidine side-chain is part of the answer and thus the dynamics of CaM would be pH dependent. This was further investigated in this thesis. Methods to carry out the project included protein expression of isotope labelled CaM-TR2C E140Q, standard protein purification and protein adapted Nuclear Magnetic Resonance (NMR) spectroscopy. The results suggest that CaM-TR2C E140Q is likely to depend on pH and that histidine 107 (H107) may have a central role in the conformational changes observed. At lower pH it was also suggested that CaM-TR2C E140Q obtained a more open conformation with weakened intramolecular interactions and that the tertiary structure of CaM-TR2C E140Q may have been disrupted.

  • 18.
    Wallenhammar, Amélie
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Anandapadmanaban, Madhanagopal
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. MRC, England.
    Lemak, Alexander
    University of Toronto, Canada; University of Toronto, Canada.
    Mirabello, Claudio
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wallner, Björn
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering.
    Sunnerhagen, Maria
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 7, article id e0181551Article in journal (Refereed)
    Abstract [en]

    Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-box and coiled-coil domains (RBCC), where the B-box domain is a unique feature of the TRIM protein family. TRIM21 is an E3 ubiquitin-protein ligase implicated in innate immune signaling by acting as an autoantigen and by modifying interferon regulatory factors. Here we report the three-dimensional solution structure of the TRIM21 B-box2 domain by nuclear magnetic resonance (NMR) spectroscopy. The structure of the B-box2 domain, comprising TRIM21 residues 86-130, consists of a short alpha-helical segment with an N-terminal short beta-strand and two anti-parallel beta-strands jointly found the core, and adopts a RING-like fold. This beta beta alpha beta core largely defines the overall fold of the TRIM21 B-box2 and the coordination of one Zn2+ ion stabilizes the tertiary structure of the protein. Using NMR titration experiments, we have identified an exposed interaction surface, a novel interaction patch where the B-box2 is likely to bind the N-terminal RING domain. Our structure together with comparisons with other TRIM B-box domains jointly reveal how its different surfaces are employed for various modular interactions, and provides extended understanding of how this domain relates to flanking domains in TRIM proteins.

  • 19.
    Yusifov, Taleh
    et al.
    Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California, Los Angeles, USA.
    Javaherian, Anoosh D.
    Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California, Los Angeles, USA.
    Pantazis, Antonios
    Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at University of California, Los Angeles, USA.
    Gandhi, Chris S.
    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, USA.
    Olcese, Olcese
    Division of Molecular Medicine, Department of Anesthesiology, Cardiovascular Research Laboratory, and Brain Research Institute, David Geffen School of Medicine at University of California, Los Angeles, USA.
    The RCK1 Domain of the Human BKCa Channel Transduces Ca2+ Binding into Structural Rearrangements2010In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 136, no 2, p. 189-202Article in journal (Refereed)
    Abstract [en]

    Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

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