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  • 1.
    Adolfsson, Dan E.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tyagi, Mohit
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Deuschmann, Adrian
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Jayaweera, Sanduni Wasana
    Lindgren, Anders E. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-β fibrils2020Ingår i: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 85, nr 21, s. 14174-14189Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    A BF3×OEt2 catalyzed intramolecular Povarov reaction was used to synthesize a library of 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with a range of O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid β fibrils in vitro. Analogs substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.

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  • 2.
    Adolfsson, Dan E.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tyagi, Mohit
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kaur, Amandeep
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bharate, Jaideep B.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Enhanement of amyloid fibril binding by ring expansion of thiazolino fused 2-pyridone peptidomimeticsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Thiazolino fused 2-pyridones undergo thiazoline ring opening by reaction with 2-nitrobenzyl bromide through thi- oether attack, and base promoted fragmentation of the resulting sulfonium ions. Subsequent deprotonation of the benzylic carbon and intramolecular 1,4-addition leads to ring closure, generating dihydrothiazine fused 2-pyridones by net ring expansion of the thiazoline ring. Application of the ring expansion procedure to the pyridine and pyrimidine fused thiazolino 2-pyridones provided compounds with enhanced fibril binding activity.

  • 3.
    Alam, Athar
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Javed, Eram
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Kumar, Rajender
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence2020Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 16, nr 4, s. 1-27, artikel-id e1008466Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Author summary Type VI secretion systems (T6SSs) are essential virulence determinants of many Gram-negative pathogens, including Francisella tularensis. This highly virulent bacterium encodes an atypical T6SS lacking ClpV, the ATPase crucial for prototypic T6SS sheath disassembly. It, however, possesses ClpB, a protein critical for heat shock survival via its interaction with DnaK. Since ClpB possesses ATPase activity, it has been hypothesized to provide a compensatory function for the absence of ClpV, a hypothesis supported by the recent findings from us and others. Here, we investigated how F. tularensis ClpB controls T6S. In silico modelling of the ClpB-DnaK complex identified key interactions that were experimentally verified. For example, mutating one of the DnaK-interacting residues rendered the bacterium exquisitely susceptible to heat shock, but had no effect on T6S and virulence. In contrast, removing the N-terminal of ClpB only had a slight effect on the heat shock response, but strongly compromised both T6S and virulence. Intriguingly, the Escherichia coli ClpB could fully complement the function of F. tularensis ClpB. The data demonstrate that the two critical roles of ClpB, mediating heat shock survival and effective T6S, are dissociated and that the N-terminal is crucial for T6S and virulence. Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the Delta clpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

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  • 4.
    Bharate, Jaideep B.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jayaweera, Sanduni Wasana
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Vielfort, Katarina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Tyagi, Mohit
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bonde, Mari
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones2021Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 19, nr 44, s. 9758-9772Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature amyloid-β and α-synuclein fibrils.

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  • 5. Bharate, Jaideep B.
    et al.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jayaweera, Sanduni Wasana
    Vielfort, Katarina
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tyagi, Mohit
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    K2S2O8-mediated aerobic oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-pyridones with aldehydes: Direct access to highly functionalized pyrimidine fused thiazolino-2-pyridones with amyloid fibril binding activity or inhibitors of Amyloid-β fibril formationManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)- thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have the ability to inhibit the formation of Amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature Amyloid-β and α-Synuclein fibrils.

  • 6.
    Bugaytsova, Jeanna A.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björnham, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik. Swedish Defence Research Agency, 906 21 Umeå, Sweden.
    Chernov, Yevgen A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gideonsson, Pär
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Henriksson, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mendez, Melissa
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sjöström, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mahdavi, Jafar
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. School of Life Sciences, CBS, University of Nottingham, NG7 2RD Nottingham, UK.
    Shevtsova, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ilver, Dag
    Moonens, Kristof
    Quintana-Hayashi, Macarena P.
    Moskalenko, Roman
    Aisenbrey, Christopher
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bylund, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Schmidt, Alexej
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Åberg, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Koeniger, Verena
    Vikström, Susanne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rakhimova, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ögren, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Avdelningen för medicin.
    Liu, Hui
    Goldman, Matthew D.
    Whitmire, Jeannette M.
    Åden, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    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 universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Holgersson, Jan
    Esberg, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Strömberg, Nicklas
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Landström, Maréne
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    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 universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Remaut, Han
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Berg, Douglas E.
    Boren, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence2017Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 21, nr 3, s. 376-389Artikel i tidskrift (Refereegranskat)
    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.

  • 7.
    Cairns, Andrew G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Vazquez-Romero, Ana
    Mahdi-Moein, Mohammad
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Elmore, Charles S.
    Takano, Akihiro
    Arakawa, Ryosuke
    Varrone, Andrea
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Schou, Magnus
    Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy2018Ingår i: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 9, nr 11, s. 2542-2547Artikel i tidskrift (Refereegranskat)
    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.

  • 8.
    Chorell, Erik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, Emma
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Evans, Margery L.
    Jain, Neha
    Götheson, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Åden, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chapman, Matthew R.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts2015Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 10, nr 10, s. 1-11, artikel-id e0140194Artikel i tidskrift (Refereegranskat)
    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.

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  • 9.
    Clifton, Luke A.
    et al.
    ISIS Neutron and Muon Source, Science and Technology Facilities Council, Didcot, United Kingdom.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wacklin-Knecht, Hanna
    European Spallation Source, European Research Infrastructure Consortium, Lund, Sweden.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Insight into Bcl-2 proteins' functioning at mitochondrial membrane level2023Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, nr 3S1, s. 232a-232a, artikel-id 1130-PosArtikel i tidskrift (Refereegranskat)
    Abstract [en]

    Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.

  • 10.
    Clifton, Luke A.
    et al.
    SIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK.
    Wacklin-Knecht, Hanna P.
    European Spallation Source ERIC, ESS, Lund, Sweden; Department of Chemistry, Division of Physical Chemistry, Lund University, Lund, Sweden.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis2023Ingår i: Science Advances, E-ISSN 2375-2548, Vol. 9, nr 22, artikel-id eadg7940Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of which were critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death. Bax initiates apoptosis by perforating mitochondrial membranes via formation of pores and extramembranous Bax-lipid complexes.

  • 11.
    Dingeldein, Artur P. G.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wacklin, Hanna P.
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Mitochondrial Membrane Organization under Oxidative Stress: Insight by Solid-State NMR and Neutron Reflectometry2019Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 116, nr 3, s. 508A-508AArtikel i tidskrift (Övrigt vetenskapligt)
  • 12.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Mikael J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Zhong, Xueyin
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy, Bochum, Germany.
    Stoll, Raphael
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy, Bochum, Germany.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies2019Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 158, s. 20-26Artikel i tidskrift (Refereegranskat)
    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.

  • 13.
    Dulko-Smith, Beata
    et al.
    Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas, USA.
    Ojeda-May, Pedro
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Högpresterande beräkningscentrum norr (HPC2N).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nam, Kwangho
    Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas, USA.
    Mechanistic basis for a connection between the catalytic step and slow opening dynamics of adenylate kinase2023Ingår i: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 63, nr 5, s. 1556-1569Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

  • 14.
    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 universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    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 universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    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 Kinase2014Ingår i: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, nr 7, s. e1003721-Artikel i tidskrift (Refereegranskat)
    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.

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  • 15. Evans, Margery L.
    et al.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Taylor, Jonathan D.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Götheson, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Li, Fei
    Koch, Marion
    Sefer, Lea
    Matthews, Steve J.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chapman, Matthew R.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation2015Ingår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 57, nr 3, s. 445-455Artikel i tidskrift (Refereegranskat)
    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.

  • 16. Jain, Neha
    et al.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nagamatsu, Kanna
    Evans, Margery L.
    Li, Xinyi
    McMichael, Brennan
    Ivanova, Magdalena I.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Buxbaum, Joel N.
    Chapman, Matthew R.
    Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin2017Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 46, s. 12184-12189Artikel i tidskrift (Refereegranskat)
    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.

  • 17. Kelly, Rachel
    et al.
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bemelmans, Alexis-Pierre
    Brouillet, Emmanuel
    Patton, Tommy
    McKernan, Declan P.
    Dowd, Eilís
    The Small Molecule Alpha-Synuclein Aggregator, FN075, Enhances Alpha-Synuclein Pathology in Subclinical AAV Rat Models2021Ingår i: Biomolecules, E-ISSN 2218-273X, Vol. 11, nr 11, artikel-id 1685Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Animal models of Parkinson’s disease, in which the human α-synuclein transgene is overexpressed in the nigrostriatal pathway using viral vectors, are widely considered to be the most relevant models of the human condition. However, although highly valid, these models have major limitations related to reliability and variability, with many animals exhibiting pronounced α-synuclein expression failing to demonstrate nigrostriatal neurodegeneration or motor dysfunction. Therefore, the aim of this study was to determine if sequential intra-nigral administration of AAV-α-synuclein followed by the small α-synuclein aggregating molecule, FN075, would enhance or precipitate the associated α-synucleinopathy, nigrostriatal pathology and motor dysfunction in subclinical models. Rats were given unilateral intra-nigral injections of AAV-α-synuclein (either wild-type or A53T mutant) followed four weeks later by a unilateral intra-nigral injection of FN075, after which they underwent behavioral testing for lateralized motor functionality until they were sacrificed for immunohistological assessment at 20 weeks after AAV administration. In line with expectations, both of the AAV vectors induced widespread overexpression of human α-synuclein in the substantia nigra and striatum. Sequential administration of FN075 significantly enhanced the α-synuclein pathology with increased density and accumulation of the pathological form of the protein phosphorylated at serine 129 (pS129-α-synuclein). However, despite this enhanced α-synuclein pathology, FN075 did not precipitate nigrostriatal degeneration or motor dysfunction in these subclinical AAV models. In conclusion, FN075 holds significant promise as an approach to enhancing the α-synuclein pathology in viral overexpression models, but further studies are required to determine if alternative administration regimes for this molecule could improve the reliability and variability in these models.

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  • 18.
    Kovermann, Michael
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural basis for catalytically restrictive dynamics of a high-energy enzyme state2015Ingår i: Nature Communications, E-ISSN 2041-1723, Vol. 6, artikel-id 7644Artikel i tidskrift (Refereegranskat)
    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.

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  • 19.
    Mikaelsson, Therese
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Johansson, Lennart B-Å
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding2013Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, nr 3, s. 694-704Artikel i tidskrift (Refereegranskat)
    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.

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    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding
  • 20.
    Mikaelsson, Therese
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Johansson, Lennart B-Å
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Macromolecular crowding effects on two homologs of ribosomal protein S16: protein-dependent structural changes and local interactions2014Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, nr 2, s. 401-410Artikel i tidskrift (Refereegranskat)
    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.

  • 21.
    Mondol, Tanumoy
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    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 ATP7B2016Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 470, nr 3, s. 663-669Artikel i tidskrift (Refereegranskat)
    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.

  • 22.
    Nadeem, Aftab
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Berg, Alexandra
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Pace, Hudson
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM).
    Alam, Athar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Toh, Eric
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zlatkov, Nikola
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Myint, Si Lhyam
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Persson, Karina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bally, Marta
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Wallenberg centrum för molekylär medicin vid Umeå universitet (WCMM). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Barandun, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae2022Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 11, artikel-id e73439Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

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  • 23.
    Nilsson, Lina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Niemiec, Moritz S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nam, Kwangho
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Computational Life Science Center (CLiC), Umeå University,.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein2013Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, nr 42, s. 13038-13050Artikel i tidskrift (Refereegranskat)
    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.

  • 24. Näsström, Thomas
    et al.
    Dahlberg, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Malyshev, Dmitry
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, Per Ola
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Karlsson, Björn C. G.
    Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts2021Ingår i: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, nr 17, artikel-id 9334Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

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  • 25. Näsström, Thomas
    et al.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Shibata, Fumina
    Andersson, Per Ola
    Karlsson, Björn C.G.
    A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations2020Ingår i: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, nr 5, artikel-id 1629Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

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  • 26. Olsen, Laura K.
    et al.
    Cairns, Andrew G.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Moriarty, Niamh
    Cabre, Silvia
    Alamilla, Veronica R.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dowd, Eilís
    McKernan, Declan P.
    Viral mimetic priming enhances α-synuclein-induced degeneration: implications for Parkinson's disease2019Ingår i: Brain, behavior, and immunity, ISSN 0889-1591, E-ISSN 1090-2139, Vol. 80, s. 525-535Artikel i tidskrift (Refereegranskat)
    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.

  • 27.
    Petzoldt, Svenja
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Center of Life and Food Sciences, Technische Universität München, Freising, Germany.
    Kahra, Dana
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kovermann, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur PG
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Niemiec, Moritz S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro2015Ingår i: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, nr 3, s. 577-585Artikel i tidskrift (Refereegranskat)
    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.

  • 28.
    Rundqvist, Louise
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olsson, Ulrika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Noncooperative folding of subdomains in Adenylate Kinase2009Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 9, s. 1911-1927Artikel i tidskrift (Refereegranskat)
    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.

  • 29.
    Singh, Pardeep
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Bartens, Christian
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pyridine-Fused 2-Pyridones via Povarov and A3 Reactions: Rapid Generation of Highly Functionalized Tricyclic Heterocycles Capable of Amyloid Fibril Binding2019Ingår i: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, nr 7, s. 3887-3903Artikel i tidskrift (Refereegranskat)
    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.

  • 30.
    Singh, Pardeep
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Cairns, Andrew G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer, Uwe H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Synthesis of Densely Functionalized N-Alkenyl 2-Pyridones via Benzyne-Induced Ring Opening of Thiazolino-Fused 2-Pyridones2019Ingår i: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 21, s. 6946-6950Artikel i tidskrift (Refereegranskat)
    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.

  • 31.
    Singh, Pardeep
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Krishnan, K Syam
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kindahl, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Synthesis of multiring fused 2‑pyridones via a nitrene insertion reaction: fluorescent modulators of α‑synuclein amyloid formation2015Ingår i: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 17, nr 24, s. 6194-6197Artikel i tidskrift (Refereegranskat)
    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.

  • 32.
    Tyagi, Mohit
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jayaweera, Sanduni Wasana
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Bharate, Jaideep B.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kiss, Anita
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sarkar, Souvik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tandem Ring Opening/Intramolecular [2 + 2] Cycloaddition Reaction for the Synthesis of Cyclobutane Fused Thiazolino-2-Pyridones2021Ingår i: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 86, nr 23, s. 16582-16592Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reaction of thiazoline fused 2-pyridones with alkyl halides in the presence of cesium carbonate opens the thiazoline ring via S-alkylation and generates N-alkenyl functionalized 2-pyridones. In the reaction with propargyl bromide, the thiazoline ring opens and subsequently closes via a [2 + 2] cycloaddition between an in situ generated allene and the α,β-unsaturated methyl ester. This method enabled the synthesis of a variety of cyclobutane fused thiazolino-2-pyridones, of which a few analogues inhibit amyloid β1–40 fibril formation. Furthermore, other analogues were able to bind mature α-synuclein and amyloid β1−40 fibrils. Several thiazoline fused 2-pyridones with biological activity tolerate this transformation, which in addition provides an exocyclic alkene as a potential handle for tuning bioactivity.

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  • 33. Tyagi, Mohit
    et al.
    Adolfsson, Dan E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Singh, Pardeep
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sarkar, Souvik
    Kiss, Anita
    Bharate, Jaideep B.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Functionalization of Thiazolino fused 2-Pyridones by thiazoline ring opening and closing: Identification of new Amyloid Binding HeterocyclesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Reaction of thiazolino fused 2-pyridones with alkyl halides in the presence of cesium carbonate opens the thiazoline ring via S-alkylation and generate N-alkenyl functionalized 2-pyridones. In the reaction with propargyl bromide, the thiazoline ring opens and subsequently closes via an intramolecular [2 + 2] cycloaddition between in situ generated allene and the α,β-unsaturated carbonyl moiety. This method enabled the synthesis of a variety of cyclobutane and thiazolino fused 2-pyridones, which were tested for α-synuclein binding activity. Most of the bioactive thiazolino fused 2-pyridones tolerated this transformation and in addition provided an exocyclic alkene as a handle for tuning bioactivity.

  • 34.
    Ul Mushtaq, Ameeq
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Alam, Athar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Backbone chemical shift assignment and dynamics of the N-terminal domain of ClpB from Francisella tularensis type VI secretion system2022Ingår i: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 16, s. 75-79Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

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  • 35.
    Ul Mushtaq, Ameeq
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ali, Katan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Domain-specific insight into the recognition of BH3-death motifs by the pro-survival Bcl-2 protein2022Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 121, nr 23, s. 4517-4525Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lymphoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, cancers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molecular basis of Bcl-2’s ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane-mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2’s functional domains engage in recognizing and inhibiting apoptotic proteins via their prominent BH3 motifs. This way, we provide a unique residue- and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

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  • 36.
    Ul Mushtaq, Ameeq
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Clifton, Luke A.
    Wacklin-Knecht, Hanna
    Campana, Mario
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Persson, Cecilia
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Neutron reflectometry and NMR spectroscopy of full-length Bcl-2 protein reveal its membrane localization and conformation2021Ingår i: Communications Biology, E-ISSN 2399-3642, Vol. 4, nr 1, artikel-id 507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis. Anti-apoptotic Bcl-2 proteins possess a hydrophobic tail-anchor enabling them to translocate to their target membrane and to shift into an active conformation where they inhibit pro-apoptotic Bcl-2 proteins to ensure cell survival. To address the unknown molecular basis of their cell-protecting functionality, we used intact human Bcl-2 protein natively residing at the mitochondrial outer membrane and applied neutron reflectometry and NMR spectroscopy. Here we show that the active full-length protein is entirely buried into its target membrane except for the regulatory flexible loop domain (FLD), which stretches into the aqueous exterior. The membrane location of Bcl-2 and its conformational state seems to be important for its cell-protecting activity, often infamously upregulated in cancers. Most likely, this situation enables the Bcl-2 protein to sequester pro-apoptotic Bcl-2 proteins at the membrane level while sensing cytosolic regulative signals via its FLD region.

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  • 37.
    Ul Mushtaq, Ameeq
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hedenström, Mattias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein - A Promising Cancer Drug Target2021Ingår i: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 26, nr 5, artikel-id 1467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

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  • 38.
    Wallgren, Marcus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pylypenko, Olena
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Mikaelsson, Therese
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Johansson, Lennart B-Å
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Rak, Alexey
    Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state2008Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, nr 4, s. 845-858Artikel i tidskrift (Refereegranskat)
    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.

  • 39.
    Ådén, Jörgen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    NMR studies of protein dynamics and structure2010Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

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    FULLTEXT02
  • 40.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P. G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-22020Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 172, artikel-id 105628Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

  • 41.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Ul Mushtaq, Ameeq
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sparrman, Tobias
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Dingeldein, Artur P.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wacklin, Hanna P.
    Wacklin, Hanna
    Clifton, Luke A.
    Gröbner, Gerhard
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Mitochondrial Membranes Involved in Apoptosis: the Bcl-2 Proteins2020Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 118, nr 3, s. 394A-394AArtikel i tidskrift (Övrigt vetenskapligt)
  • 42.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Verma, Abhinav
    Schug, Alexander
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity2012Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, nr 40, s. 16562-16570Artikel i tidskrift (Refereegranskat)
    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.

  • 43.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Storm, Patrik
    Weise, Christoph
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Christiansen, Alexander
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Schröder, Wolfgang
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Funk, Christiane
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Arabidopsis thaliana peroxiredoxin Q is extraordinarily dynamic on the μs-ms timescaleManuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 44.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wallgren, Marcus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Storm, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Weise, Christoph
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Christiansen, Alexander
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Schröder, Wolfgang P
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Funk, Christiane
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Extraordinary μs-ms backbone dynamics in Arabidopsis thaliana peroxiredoxin Q2011Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1814, nr 12, s. 1880-1890Artikel i tidskrift (Refereegranskat)
    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.

  • 45.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Weise, Christoph
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Brännström, Kristoffer
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structural topology and activation of an initial adenylate kinase-substrate complex2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 6, s. 1055-1061Artikel i tidskrift (Refereegranskat)
    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.

  • 46.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Folding of an unfolded protein by macromolecular crowding in vitro2014Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 14, s. 2271-2277Artikel i tidskrift (Refereegranskat)
    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.

  • 47.
    Ådén, Jörgen
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wolf-Watz, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    NMR identification of transient complexes critical to adenylate kinase catalysis2007Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, nr 45, s. 14003-12Artikel i tidskrift (Refereegranskat)
    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.

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