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  • 1.
    Bugaytsova, Jeanna A.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Björnham, Oscar
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics. Swedish Defence Research Agency, 906 21 Umeå, Sweden.
    Chernov, Yevgen A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gideonsson, Pär
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mendez, Melissa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sjöström, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mahdavi, Jafar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. School of Life Sciences, CBS, University of Nottingham, NG7 2RD Nottingham, UK.
    Shevtsova, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ilver, Dag
    Moonens, Kristof
    Quintana-Hayashi, Macarena P.
    Moskalenko, Roman
    Aisenbrey, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schmidt, Alexej
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Åberg, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Koeniger, Verena
    Vikström, Susanne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ögren, Johan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Section of Medicine.
    Liu, Hui
    Goldman, Matthew D.
    Whitmire, Jeannette M.
    Åden, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Younson, Justine
    Kelly, Charles G.
    Gilman, Robert H.
    Chowdhury, Abhijit
    Mukhopadhyay, Asish K.
    Nair, G. Balakrish
    Papadakos, Konstantinos S.
    Martinez-Gonzalez, Beatriz
    Sgouras, Dionyssios N.
    Engstrand, Lars
    Unemo, Magnus
    Danielsson, Dan
    Suerbaum, Sebastian
    Oscarson, Stefan
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holgersson, Jan
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Landström, Maréne
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Eldridge, Angela M.
    Chromy, Brett A.
    Hansen, Lori M.
    Solnick, Jay V.
    Linden, Sara K.
    Haas, Rainer
    Dubois, Andre
    Merrell, D. Scott
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Remaut, Han
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berg, Douglas E.
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence2017In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, no 3, p. 376-389Article in journal (Refereed)
    Abstract [en]

    The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

  • 2.
    Cairns, Andrew G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vazquez-Romero, Ana
    Mahdi-Moein, Mohammad
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Elmore, Charles S.
    Takano, Akihiro
    Arakawa, Ryosuke
    Varrone, Andrea
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schou, Magnus
    Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy2018In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 9, no 11, p. 2542-2547Article in journal (Refereed)
    Abstract [en]

    Previous work in our laboratories has identified a series of peptidomimetic 2-pyridone molecules as modulators of alpha-synuclein (α-syn) fibrillization in vitro. As a first step toward developing molecules from this scaffold as positron emission tomography imaging agents, we were interested in evaluating their blood-brain barrier permeability in nonhuman primates (NHP) in vivo. For this purpose, 2-pyridone 12 was prepared and found to accelerate α-syn fibrillization in vitro. Acid 12, and its acetoxymethyl ester analogue 14, were then radiolabeled with 11C (t1/2 = 20.4 min) at high radiochemical purity (>99%) and high specific radioactivity (>37 GBq/μmol). Following intravenous injection of each compound in NHP, a 4-fold higher radioactivity in brain was observed for [11C]14 compared to [11C]12 (0.8 vs 0.2 SUV, respectively). [11C]14 was rapidly eliminated from plasma, with [11C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein deposits in brain.

  • 3.
    Chorell, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Emma
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Evans, Margery L.
    Jain, Neha
    Götheson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Åden, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chapman, Matthew R.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 10, p. 1-11, article id e0140194Article in journal (Refereed)
    Abstract [en]

    Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.

  • 4.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zhong, Xueyin
    Stoll, Raphael
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bax to the future – A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies2019In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, p. 20-26Article in journal (Refereed)
    Abstract [en]

    Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

  • 5.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wacklin, Hanna P.
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry2019In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, no 3, p. 508A-508AArticle in journal (Other academic)
  • 6.
    Esteban-Martin, Santiago
    et al.
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center - BSC, Barcelona, Spain.
    Fenwick, Robert Bryn
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine – IRB Barcelona, Barcelona, Spain.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cossins, Benjamin
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center - BSC, Barcelona, Spain.
    Bertoncini, Carlos W.
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine – IRB Barcelona, Barcelona, Spain.
    Guallar, Victor
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center - BSC, Barcelona, Spain.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Salvatella, Xavier
    Joint BSC-CRG-IRB Research Programme in Computational Biology, Institute for Research in Biomedicine – IRB Barcelona, Barcelona, Spain.
    Correlated Inter-Domain Motions in Adenylate Kinase2014In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, no 7, p. e1003721-Article in journal (Refereed)
    Abstract [en]

    Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

  • 7. Evans, Margery L.
    et al.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Taylor, Jonathan D.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Götheson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Li, Fei
    Koch, Marion
    Sefer, Lea
    Matthews, Steve J.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chapman, Matthew R.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation2015In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 57, no 3, p. 445-455Article in journal (Refereed)
    Abstract [en]

    Summary Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

  • 8. Jain, Neha
    et al.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nagamatsu, Kanna
    Evans, Margery L.
    Li, Xinyi
    McMichael, Brennan
    Ivanova, Magdalena I.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Buxbaum, Joel N.
    Chapman, Matthew R.
    Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 46, p. 12184-12189Article in journal (Refereed)
    Abstract [en]

    During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer’s-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.

  • 9.
    Kovermann, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural basis for catalytically restrictive dynamics of a high-energy enzyme state2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7644Article in journal (Refereed)
    Abstract [en]

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.

  • 10.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 3, p. 694-704Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

  • 11.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding effects on two homologs of ribosomal protein S16: protein-dependent structural changes and local interactions2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 2, p. 401-410Article in journal (Refereed)
    Abstract [en]

    Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16T(herme)) and mesophilic (S161homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic Meso, protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Forster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16 Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16me, allowing measurements of three intraprotein distances. All S16meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two 516-rhermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

  • 12.
    Mondol, Tanumoy
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Biology and Biological Engineering Department, Chalmers University of Technology, Gothenburg, Sweden.
    Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B2016In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 470, no 3, p. 663-669Article in journal (Refereed)
    Abstract [en]

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

  • 13.
    Nilsson, Lina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nam, Kwangho
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Computational Life Science Center (CLiC), Umeå University,.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 42, p. 13038-13050Article in journal (Refereed)
    Abstract [en]

    Although strictly regulated, pH and solute concentrations in cells may exhibit temporal and spatial fluctuations. Here we study the effect of such changes on the stability, structure, and dynamics in vitro and in silico of a two-domain construct (WD56) of the fifth and sixth metal-binding domains of the copper transport protein, ATP7B (Wilson disease protein). We find that the thermal stability of WD56 is increased by 40 °C when increasing the pH from 5.0 to 7.5. In contrast, addition of salt at pH 7.2 decreases WD56 stability by up to 30 °C. In agreement with domain-domain coupling, fractional copper loading increases the stability of both domains. HSQC chemical shift changes demonstrate that, upon lowering the pH from 7.2 to 6, both His in WD6 as well as the second Cys of the copper site in each domain become protonated. MD simulations reveal increased domain-domain fluctuations at pH 6 and in the presence of high salt concentration, as compared to at pH 7 and low salt concentration. Thus, the surface charge distribution at high pH contributes favorably to overall WD56 stability. By introducing more positive charges by lowering the pH, or by diminishing charge-charge interactions by salt, fluctuations among the domains are increased and thereby overall stability is reduced. Copper transfer activity also depends on pH: delivery of copper from chaperone Atox1 to WD56 is more efficient at pH 7.2 than at pH 6 by a factor of 30. It appears that WD56 is an example where the free energy landscapes for folding and function are linked via structural stability.

  • 14. Olsen, Laura K.
    et al.
    Cairns, Andrew G.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Moriarty, Niamh
    Cabre, Silvia
    Alamilla, Veronica R.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dowd, Eilís
    McKernan, Declan P.
    Viral mimetic priming enhances α-synuclein-induced degeneration: implications for Parkinson's disease2019In: Brain, behavior, and immunity, ISSN 0889-1591, E-ISSN 1090-2139, Vol. 80, p. 525-535Article in journal (Refereed)
    Abstract [en]

    Evidence is accumulating to suggest that viral infections and consequent viral-mediated neuroinflammation may contribute to the etiology of idiopathic Parkinson’s disease. Moreover, viruses have been shown to influence α-synuclein oligomerization as well as the autophagic clearance of abnormal intra-cellular proteins aggregations, both of which are key neuropathological events in Parkinson’s disease pathogenesis. To further investigate the interaction between viral-mediated neuroinflammation and α-synuclein aggregation in the context of Parkinson’s disease, this study sought to determine the impact of viral neuroinflammatory priming on α-synuclein aggregate-induced neuroinflammation and neurotoxicity in the rat nigrostriatal pathway. To do so, male Sprague-Dawley rats were intra-nigrally injected with a synthetic mimetic of viral dsRNA (poly I:C) followed two weeks later by a peptidomimetic small molecule which accelerates α-synuclein fibril formation (FN075). The impact of the viral priming on α-synuclein aggregation-induced neuroinflammation, neurodegeneration and motor dysfunction was assessed. We found that prior administration of the viral mimetic poly I:C significantly exacerbated or precipitated the α-synuclein aggregate induced neuropathological and behavioral effects. Specifically, sequential exposure to the two challenges caused a significant increase in nigral microgliosis (p < 0.001) and astrocytosis (p < 0.01); precipitated a significant degeneration of the nigrostriatal cell bodies (p < 0.05); and precipitated a significant impairment in forelimb kinesis (p < 0.01) and sensorimotor integration (p < 0.01). The enhanced sensitivity of the nigrostriatal neurons to pathological α-synuclein aggregation after viral neuroinflammatory priming further suggests that viral infections may contribute to the etiology and pathogenesis of Parkinson’s disease.

  • 15.
    Petzoldt, Svenja
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Center of Life and Food Sciences, Technische Universität München, Freising, Germany.
    Kahra, Dana
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur PG
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro2015In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, p. 577-585Article in journal (Refereed)
    Abstract [en]

    After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P-1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

  • 16.
    Rundqvist, Louise
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olsson, Ulrika
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Noncooperative folding of subdomains in Adenylate Kinase2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 9, p. 1911-1927Article in journal (Refereed)
    Abstract [en]

    Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and Cα chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.

  • 17.
    Singh, Pardeep
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Adolfsson, Dan E.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cairns, Andrew G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bartens, Christian
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pyridine-Fused 2-Pyridones via Povarov and A3 Reactions: Rapid Generation of Highly Functionalized Tricyclic Heterocycles Capable of Amyloid Fibril Binding2019In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, no 7, p. 3887-3903Article in journal (Refereed)
    Abstract [en]

    We here describe the use of three-component reactions to synthesize tricyclic pyridine ring-fused 2-pyridones. The developed protocols have a wide substrate scope and allow for the installation of diverse chemical functionalities on the tricyclic central fragment. Several of these pyridine-fused rigid polyheterocycles are shown to bind to Aβ and α-synuclein fibrils, which are associated with neurodegenerative diseases.

  • 18.
    Singh, Pardeep
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cairns, Andrew G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Adolfsson, Dan E.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Synthesis of Densely Functionalized N-Alkenyl 2-Pyridones via Benzyne-Induced Ring Opening of Thiazolino-Fused 2-Pyridones2019In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052Article in journal (Refereed)
    Abstract [en]

    We report the synthesis of 6-arylthio-substituted-N-alkenyl 2-pyridones by ring opening of bicyclic thiazolino-2-pyridones with arynes. Varied functionalization was used to investigate scope and substituent influences on reactivity. Selected conditions favor thioether ring opening over [4 + 2] cycloaddition and an unusual aryne incorporating ring expansion. Deuterium labeling was used to clarify observed reactivity. Using the knowledge, we produced drug-like molecules with complex substitution patterns and show how thioether ring opening can be used on scaffolds with competing reactivities.

  • 19.
    Singh, Pardeep
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Krishnan, K Syam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kindahl, Tomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Synthesis of multiring fused 2‑pyridones via a nitrene insertion reaction: fluorescent modulators of α‑synuclein amyloid formation2015In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 17, no 24, p. 6194-6197Article in journal (Refereed)
    Abstract [en]

    An efficient, straightforward method for the synthesis of thiazolo-2-pyridone embedded peptidomimetic polyheterocycles via a catalyst-free, microwave-assisted, intramolecular C−H amination reaction is reported. All the synthesized polyheterocycles were evaluated for their fluorescent properties and effect on α-synuclein amyloid formation.

  • 20.
    Wallgren, Marcus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pylypenko, Olena
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Mikaelsson, Therese
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rak, Alexey
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 4, p. 845-858Article in journal (Refereed)
    Abstract [en]

    Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16meso) and hyperthermophilic (S16thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔCp0) of 6.4 and 3.3 kJ mol− 1 K− 1, respectively. A strong linear correlation between ΔCp0 and melting temperature (Tm) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16meso folds through an apparent two-state model, whereas S16thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16thermo is more compact relative to S16meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16thermo that appears to account for the large difference in quantified ΔCp0 values and, in turn, parts of the observed extreme thermal stability of S16thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.

  • 21.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    NMR studies of protein dynamics and structure2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.

  • 22.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Verma, Abhinav
    Schug, Alexander
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 40, p. 16562-16570Article in journal (Refereed)
    Abstract [en]

    Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AKeco) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turn-over rate is inversely proportional to the life-time of the active conformation. Therefore, structural plasticity is intimately cou-pled to enzymatic turn-over in AKeco. Here we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate, and in turn, the turn-over number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding sub-domains results in a population shift towards the open conformation and a resulting increase of kcat. Addition of the osmolyte TMAO to AKeco results in population shift towards the closed conformation and a significant reduction of kcat. The Michaelis constants (KM) scale with the change in kcat, which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, kcat and KM are mutually dependent and, in the case of AKeco, any perturbation that modulates kcat is mirrored with a proportional response in KM. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affect enzymatic catalysis. From an evolutionary perspective our findings suggests that, for AKeco, there exists ample flexibility to obtain a specificity constant (kcat/KM) that commensurate with the exerted cellular selective pressure.

  • 23.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Storm, Patrik
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schröder, Wolfgang
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Arabidopsis thaliana peroxiredoxin Q is extraordinarily dynamic on the μs-ms timescaleManuscript (preprint) (Other academic)
    Abstract [en]

    Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted as the resolving cysteine, generating an intermolecular disulphide bond to expel a second water molecule into solution. PrxQ is finally recycled to the active state by a thioredoxin dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here we have performed a detailed study of the structure and dynamics of PrxQ in both the oxidized and reduced state. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Model-free analyses of NMR spin relaxation show that PrxQ is monomeric in both oxidation states. As evident from fast R2 relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs-ms timescale. The ground state of the conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.

  • 24.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wallgren, Marcus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Storm, Patrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schröder, Wolfgang P
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Extraordinary μs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1814, no 12, p. 1880-1890Article in journal (Refereed)
    Abstract [en]

    Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin-dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted the resolving cysteine, generating an intramolecular disulfide bond and release of a second water molecule. PrxQ is recycled to the active state by a thioredoxin-dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here, we have performed a detailed study of the activity, structure and dynamics of PrxQ in both the oxidized and reduced states. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Analysis of NMR spin relaxation rates shows that PrxQ is monomeric in both oxidized and reduced states. As evident from R(2) relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs-ms timescale. The ground state of this conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.

  • 25.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 6, p. 1055-1061Article in journal (Refereed)
    Abstract [en]

    Enzymatic activity is ultimately defined by the structure, chemistry and dynamics of the Michaelis complex. There exist a large number of experimentally determined structures between enzymes and substrates or substrate analogues or inhibitors. However, transient, short-lived encounter and equilibrium structures also play fundamental roles during enzymatic reaction cycles. Such structures are inherently difficult to study with conventional experimental techniques. The enzyme adenylate kinase undergoes major conformational rearrangements in response to binding of its substrates ATP and AMP. ATP is sandwiched between two binding surfaces in the closed and active enzyme conformation. Thus, ade-nylate kinase harbors two spatially distant surfaces in the substrate free open conformation of which one is responsible for the initial interaction with ATP. Here, we have performed primarily nuclear magnetic resonance experiments on Escherichia coli adenylate kinase (AKeco) variants that enabled identification of the site responsible for the initial ATP interaction. This allowed a characterization of the structural topology of an initial equilibrium complex between AKeco and ATP. Based on the results it is suggested that the ATP binding mechanism to AKeco is a mixture between "induced fit" and "conformational selection" models. It is shown that ATP is activated in the initial enzyme bound complex since it displays an appreciable rate of non-productive ATP hydrolysis. In summary our results provide novel structural and functional insights into adenylate kinase catalysis.

  • 26.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Folding of an unfolded protein by macromolecular crowding in vitro2014In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, no 14, p. 2271-2277Article in journal (Refereed)
    Abstract [en]

    Protein folding in vivo takes place in a highly crowded environment. The resulting excluded volume forces are thought to stabilize folded forms of proteins. In agreement, many in vitro studies have shown that the presence of macromolecular crowding agents increases the stability of folded proteins but often by only a few kJ per mol. Although it should not matter at what position in the transition between folded and unfolded forms the effect of crowding is employed, there have been no studies assessing whether excluded volume forces alone can correctly fold polypeptides that are mostly unfolded. However, some studies have indicated that the effect of crowding becomes larger the more destabilized the protein is (but still being folded), suggesting that the crowding effect may be exaggerated for unfolded proteins. To address this question directly, we turned to a destabilized mutant of protein L that is mostly unfolded in water but can be folded upon addition of salt. We find that the effect of 200 mg/mL Dextran 20 on the folding equilibrium constant for unfolded protein L (ΔΔGU ≈ 2 kJ mol(-1)) matches the crowding effects found on the folded wild type protein and the mutant when prefolded by salt. This result indicates that the excluded volume effect is independent of starting protein stability and that crowding can shift the reaction toward the folded form when the polypeptide is in the transition region between folded and unfolded states.

  • 27.
    Ådén, Jörgen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    NMR identification of transient complexes critical to adenylate kinase catalysis2007In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 45, p. 14003-12Article in journal (Refereed)
    Abstract [en]

    A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.

1 - 27 of 27
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