umu.sePublications
Change search
Refine search result
12 1 - 50 of 69
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Aguilar, Ximena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding extended to a heptameric system: the co-chaperonin protein 102011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 14, p. 3034-3044Article in journal (Refereed)
    Abstract [en]

    Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.

  • 2.
    Andersson, C David
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Karlberg, Tobias
    Ekblad, Torun
    Lindgren, Anders E G
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Thorsell, Ann-Gerd
    Spjut, Sara
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Uciechowska, Urszula
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weigelt, Johan
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schüler, Herwig
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Discovery of Ligands for ADP-Ribosyltransferases via Docking-Based Virtual Screening2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 17, p. 7706-7718Article in journal (Refereed)
    Abstract [en]

    The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyses the transfer of ADP-ribose units onto substrate proteins, using nicotinamide adenine dinucleotide (NAD(+)) as a co-substrate. They have a documented role in chromatin remodelling and DNA repair; and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. Using virtual screening we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.

  • 3.
    Andersson, Emma K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Evans, Margery L.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Anders E.G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hufnagel, David A.
    Bhattacharya, Moumita
    Tessier, Peter M.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chapman, Matthew R.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). University of Michigan, USA.
    Modulation of Curli Assembly and Pellicle Biofilm Formation by Chemical and Protein Chaperones2013In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 20, no 10, p. 1245-1254Article in journal (Refereed)
    Abstract [en]

    Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by "chemical chaperones."

  • 4.
    Berg, Lotta
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Qian, Weixing
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, C. David
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ekström, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Similar but Different: Thermodynamic and Structural Characterization of a Pair of Enantiomers Binding to Acetylcholinesterase2012In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 51, no 51, p. 12716-12720Article in journal (Refereed)
    Abstract [en]

    Take a closer look: Unexpectedly, a pair of enantiomeric ligands proved to have similar binding affinities for acetylcholinesterase. Further studies indicated that the enantiomers exhibit different thermodynamic profiles. Analyses of the noncovalent interactions in the protein-ligand complexes revealed that these differences are partly due to nonclassical hydrogen bonds between the ligands and aromatic side chains of the protein.

  • 5.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Aguilar, Ximena
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rautio, Linn
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 13, p. 5938-5950Article in journal (Refereed)
    Abstract [en]

    Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

  • 6.
    Chen, Eefei
    et al.
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Kliger, David S
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, p. 9836-9845Article in journal (Refereed)
    Abstract [en]

    Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

  • 7. Chen, Mingzhi
    et al.
    Dousis, Athanasios D
    Wuc, Yinghao
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Ma, Jianpeng
    Predicting Protein Folding Cores by Empirical Potential Functions2009In: Archives of Biochemistry and Biophysics, Vol. 483, no 1, p. 16-22Article in journal (Refereed)
    Abstract [en]

    Theoretical and in vitro experiments suggest that protein-folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14, 1401 (2006)), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.

  • 8.
    Chermenina, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Histology and Cell Biology.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Henrik, Antti
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Strömberg, Ingrid
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Histology and Cell Biology.
    A novel animal model for Parkinson's disease based on in vivo effects of small-molecule of alpha-synucleinManuscript (preprint) (Other academic)
    Abstract [en]

    Amyloid fibrils of alpha-synuclein are major constituents of Lewy bodies, the pathological hallmark of Parkinson’s disease. Monomeric α-synuclein is involved in synaptic vesicle trafficking and long-term maintenance of neurons. The underlying mechanisms of Parkinson’s disease are not known but it has been proposed that oligomers of α-synuclein, formed during the aggregation process, are toxic to neurons. To search for a new animal model of Parkinson’s disease, here we capitalized on the in vitro discovery of a small-molecule templator of α-synuclein fibrillization, the 2-pyridone, FN075. FN075 and MS382, another 2-pyridone variant that act as an inhibitor of amyloids in vitro, were injected into the striatum or substantia nigra of normal C57Bl/6 mice. No acute toxicity of the compounds was detected, as there was 100 % survival of the injected mice. At 6 months after the striatal injection, sensorimotor functions were impaired with no reduction in TH-positive neurons in the substantia nigra in mice injected with FN075, whereas mice injected with MS382 or vehicle had no dysfunctions. Injection of FN075 into the substantia nigra revealed a significant loss of TH-positive neurons already at 3 months and TH-negative inclusion-like structures were detected in substantia nigra neurons of these mice. Thus, the results suggest that injection of a templator of α-synuclein aggregation into the brain of normal mice can serve as a novel experimental design for an animal model of Parkinson’s disease.

  • 9.
    Chermenina, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokrzywa, Malgorzata
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Strömberg, Ingrid
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Single injection of small-molecule amyloid accelerator results in cell death of nigral dopamine neurons in mice2015In: Parkinson's Disease, ISSN 2090-8083, E-ISSN 2042-0080, Vol. 1, article id 15024Article in journal (Refereed)
    Abstract [en]

    The assembly process of a-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson´s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of a-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of a-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and follwed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substansia nigra of normal and a-synuclein knockout mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed singificant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in a-synuclein knock-out mice injected with accelerator intor the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson´s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

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

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

  • 11.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas, USA .
    Samiotakis, Antonios
    Department of Physics, University of Houston, Houston, Texas, USA .
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas, USA .
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Factors defining effects of Macromolecular crowding on protein stability: an in vitro/in silico case study using cytochrome c2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 31, p. 6519-6530Article in journal (Refereed)
    Abstract [en]

    Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.

  • 12.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, no 7, p. 1689-1699Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.

  • 13.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Synthetic crowding agent dextran causes excluded volume interactions exclusively to tracer protein apoazurin2014In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 5, p. 811-814Article in journal (Refereed)
    Abstract [en]

    To understand protein biophysics in crowded cellular environments, researchers often use synthetic polymers as 'crowding agents' in vitro. The idea is that these agents will occupy space and reproduce the in vivo scenario in terms of excluded volume. However, recent work has challenged this concept and pointed out that attractive interactions between protein and crowding agent will provide an enthalpic contribution to the overall effect on protein thermodynamics. Here we use a typical synthetic crowding agent and a well-studied model protein to demonstrate in a window of 50 K that the presence of dextran 20 affects apoazurin by steric repulsion. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

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

  • 15. Gomes, Claudio M
    et al.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Protein folding and metal ions: mechanisms, biology and disease2010Book (Other academic)
    Abstract [en]

    The role of metal ions in protein folding and structure is a critical topic to a range of scientists in numerous fields, particularly those working in structural biology and bioinorganic chemistry, those studying protein folding and disease, and those involved in the molecular and cellular aspects of metals in biological systems. Protein Folding and Metal Ions: Mechanisms, Biology and Disease presents the contributions of a cadre of international experts who offer a comprehensive exploration of this timely subject at the forefront of current research. Divided into four sections, this volume: Provides case study examples of protein folding and stability studies in particular systems or proteins that comprise different metal ions of co-factors Reviews the proteins that shuttle metal ions in the cell to a particular target metalloprotein Illustrates how metal binding can be connected to pathological protein conformations in unrelated diseases, from cancer to protein deposition disorders such as Parkinson's disease Addresses protein redesign of metal-containing proteins by computational methods, folding simulation studies, and work on model peptides -- dissecting the relative energetic contribution of metals sites to protein folding and stability Together, the 13 chapters in this text cogently describe the state of the science today, illuminate current challenges, propose future possibilities, and encourage further study in this area that offers much promise especially with regard to novel approaches to the treatment of some of the most challenging and tragic diseases.  

  • 16.
    Good, James A. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Christopher
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hansen, Sabine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wall, Jessica
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Krishnan, Syam
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Niemiec, Moritz Sebastian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer–Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA2016In: Cell chemical biology, ISSN 2451-9448, Vol. 23, no 3, p. 404-414Article in journal (Refereed)
    Abstract [en]

    The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

  • 17. Guelker, Megan
    et al.
    Stagg, Loren
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Shamoo, Yousif
    Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin2009In: Acta crystallographica. Section D, Biological crystallography, ISSN 1399-0047, Vol. 65, no Pt 6, p. 523-34Article in journal (Refereed)
    Abstract [en]

    The crystal structure of oxidized flavodoxin from Desulfovibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3(1)21 and P4(3), at 2.5 and 2.0 A resolution, respectively. Structure determination in space group P3(1)21 was challenging owing to the presence of pseudo-translational symmetry and a high copy number in the asymmetric unit (8). Initial phasing attempts in space group P3(1)21 by molecular replacement using a poor search model (46% identity) and multi-wavelength anomalous dispersion were unsuccessful. It was necessary to solve the structure in a second crystal form, space group P4(3), which was characterized by almost perfect twinning, in order to obtain a suitable search model for molecular replacement. This search model with complementary approaches to molecular replacement utilizing the pseudo-translational symmetry operators determined by analysis of the native Patterson map facilitated the selection and manual placement of molecules to generate an initial solution in the P3(1)21 crystal form. During the early stages of refinement, application of the appropriate twin law, (-h, -k, l), was required to converge to reasonable R-factor values despite the fact that in the final analysis the data were untwinned and the twin law could subsequently be removed. The approaches used in structure determination and refinement may be applicable to other crystal structures characterized by these complicating factors. The refined model shows flexibility of the flavin mononucleotide coordinating loops indicated by the isolation of two loop conformations and provides a starting point for the elucidation of the mechanism used for protein-partner recognition.

  • 18. Homouz, Dirar M
    et al.
    Perham, Michael
    Samiotakis, Antonios
    Cheung, Margaret
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Crowded, cell-like environment induces shape changes in aspherical protein2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 96, no 3, Supplement 1, p. 568a-Article in journal (Refereed)
    Abstract [en]

    Protein dynamics in cells may be different from that in dilute solutions in vitro since the environment in cells is highly concentrated with other macromolecules. This volume exclusion due to macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, here we have investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents using in silico and in vitro approaches. By coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fraction of crowding agents (fc) as well as of crowding agent geometry (sphere or spherocylinder) at high fc. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we have identified in silico crowding conditions that best enhance protein stability and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. The test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. We propose that macromolecular crowding effects may be a tool for manipulation of protein folding and function in living cells.

  • 19. Homouz, Dirar
    et al.
    Perham, Michael
    Samiotakis, Antonios
    Cheung, Margaret S
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Crowded, cell-like environment induces shape changes in aspherical protein2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 33, p. 11754-11759Article in journal (Refereed)
    Abstract [en]

    How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein–protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region. Our work demonstrates the malleability of “native” proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo.

  • 20. Homouz, Dirar
    et al.
    Stagg, Loren
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Cheung, Margaret S
    Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin2009In: Biophysical Journal, Vol. 96, no 2, p. 671-80Article in journal (Refereed)
    Abstract [en]

    Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (c) as well as of crowding agent geometry (sphere or spherocylinder) at high c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.

  • 21.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nordvall, Lina-Maria
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Golla, Krishna Prasad
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: templation and inhibition involve oligomers with different structure2013In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 532, no 2, p. 84-90Article in journal (Refereed)
    Abstract [en]

    In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation.

  • 22.
    Horvath, Istvan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph F
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Emma K
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hultgren, Scott J
    Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, United States.
    Chapman, Matthew
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 7, p. 3439-3444Article in journal (Refereed)
    Abstract [en]

    Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.

  • 23. Hussain, Faiza
    et al.
    Olson, John S
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Conserved residues modulate copper release in human copper chaperone Atox12008In: Proceedings of the National Academy of Sciences of the United States of America PNAS, Vol. 105, no 32, p. 11158-63Article in journal (Refereed)
    Abstract [en]

    It is unclear how the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson and Menkes disease proteins in the cytoplasm. To begin to address this problem, we have characterized Cu(I) release from wild-type Atox1 and two point mutants (Met10Ala and Lys60Ala). The dynamics of Cu(I) displacement from holo-Atox1 were measured by using the Cu(I) chelator bicinchonic acid (BCA) as a metal acceptor. BCA removes Cu(I) from Atox1 in a three-step process involving the bimolecular formation of an initial Atox1–Cu–BCA complex followed by dissociation of Atox1 and the binding of a second BCA to generate apo-Atox1 and Cu–BCA2. Both mutants lose Cu(I) more readily than wild-type Atox1 because of more rapid and facile displacement of the protein from the Atox1–Cu–BCA intermediate by the second BCA. Remarkably, Cu(I) uptake from solution by BCA is much slower than the transfer from holo-Atox1, presumably because of slow dissociation of DTT–Cu complexes. These results suggest that Cu chaperones play a key role in making Cu(I) rapidly accessible to substrates and that the activated protein–metal–chelator complex may kinetically mimic the ternary chaperone–metal–target complex involved in Cu(I) transfer in vivo.

  • 24. Hussain, Faiza
    et al.
    Rodriguez-Granillo, Agustina
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lysine-60 in Copper Chaperone Atox1 Plays an Essential Role in Adduct Formation with a Target Wilson Disease Domain2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 45, p. 16371-3Article in journal (Refereed)
    Abstract [en]

    The mechanism by which the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson disease (WD) protein for insertion into cuproenzymes is unclear. Using near-UV circular dichroism as a new tool to probe chaperone-target interactions, in combination with gel filtration and molecular dynamics simulations, we here demonstrate that Atox1 forms a stable Cu-dependent adduct with the fourth metal-binding domain of WD (WD4). Using point-mutated Atox1 variants, we show that the adduct forms in the absence of conserved residues M10 or T11 but K60 is essential for heterocomplex formation and Cu transfer. Dissection of heterocomplex energetic components reveals a crucial role for K60-mediated electrostatic interaction.

  • 25.
    Kahra, Dana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemistry Department, University of Konstanz, Konstanz, Germany.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox12016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 1, p. 95-102Article in journal (Refereed)
    Abstract [en]

    Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, K-D, of similar to 10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

  • 26.
    Kahra, Dana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mondol, Tanumoy
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro2015In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 22, no 6, p. 532-538Article in journal (Refereed)
    Abstract [en]

    After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

  • 27. Maxwell, Karen L.
    et al.
    Wildes, David
    Zarrine-Afsar, Arash
    De Los Rios, Miguel A.
    Brown, Andrew G.
    Friel, Claire T.
    Hedberg, Linda
    Horng, Jia-Cherng
    Bona, Diane
    Miller, Erik J.
    Vallée-Bélisle, Alexis
    Main, Ewan R.G.
    Bemporad, Francesco
    Qiu, Linlin
    Teilum, Kaare
    Vu, Ngoc-Diep
    Edwards, Aled M.
    Ruczinski, Ingo
    Poulsen, Flemming M.
    Kragelund, Birthe B.
    Michnick, Stephen W.
    Chiti, Fabrizio
    Bai, Yawen
    Hagen, Stephen J.
    Serrano, Luis
    Oliveberg, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry.
    Raleigh, Daniel P.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Radford, Sheena E.
    Jackson, Sophie E.
    Sosnick, Tobin R.
    Marqusee, Susan
    Davidson, Alan R.
    Plaxco, Kevin W.
    Protein folding: Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins2005In: Protein Science, Vol. 14, p. 602-16Article in journal (Refereed)
    Abstract [en]

    Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

  • 28.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 3, p. 694-704Article in journal (Refereed)
    Abstract [en]

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

  • 29.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding effects on two homologs of ribosomal protein S16: protein-dependent structural changes and local interactions2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 2, p. 401-410Article in journal (Refereed)
    Abstract [en]

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

  • 30. Miller, Corwin
    et al.
    Davlieva, Milya
    Wilson, Corey
    White, Kristopher I
    Couñago, Rafael
    Wu, Gang
    Myers, Jeffrey C
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shamoo, Yousif
    Experimental evolution of adenylate kinase reveals contrasting strategies toward protein thermostability2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 3, p. 887-896Article in journal (Refereed)
    Abstract [en]

    Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.

  • 31.
    Mondol, Tanumoy
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Biology and Biological Engineering Department, Chalmers University of Technology, Gothenburg, Sweden.
    Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B2016In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 470, no 3, p. 663-669Article in journal (Refereed)
    Abstract [en]

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

  • 32. Neumann, Oara
    et al.
    Zhang, Dongmao
    Tam, Felicia
    Lal, Surbhi
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Halas, Naomi J
    Direct optical detection of aptamer conformational changes induced by target molecules2009In: Analytical chemistry, ISSN 1520-6882, Vol. 81, no 24, p. 10002-6Article in journal (Refereed)
    Abstract [en]

    Aptamers are single-stranded DNA/RNA oligomers that fold into three-dimensional conformations in the presence of specific molecular targets. Surface-enhanced Raman spectroscopy (SERS) of thiol-bound DNA aptamer self-assembled monolayers on Au nanoshell surfaces provides a direct, label-free detection method for the interaction of DNA aptamers with target molecules. A spectral cross-correlation function, Gamma, is shown to be a useful metric to quantify complex changes in the SERS spectra resulting from conformational changes in the aptamer induced by target analytes. While the pristine, unexposed anti-PDGF (PDGF = platelet-derived growth factor) aptamer yields highly reproducible spectra with Gamma = 0.91 +/- 0.01, following incubation with PDGF, the reproducibility of the SERS spectra is dramatically reduced, yielding Gamma = 0.67 +/- 0.02. This approach also allows us to discriminate the response of a cocaine aptamer to its target from its weaker response to nonspecific analyte molecules.

  • 33.
    Niemiec, Moritz S.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur P. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Enthalpy-entropy compensation at play in human copper ion transfer2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 10518Article in journal (Refereed)
    Abstract [en]

    Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C-1 but not C-2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.

  • 34.
    Niemiec, Moritz S.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur P. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 6, p. 1037-1047Article in journal (Refereed)
    Abstract [en]

    To avoid toxicity and control levels of metal ions, organisms have developed specific metal transport systems. In humans, the cytoplasmic Cu chaperone Atox1 delivers Cu to metal-binding domains of ATP7A/B in the Golgi, for incorporation into Cu-dependent proteins. The Cu-binding motif in Atox1, as well as in target Cu-binding domains of ATP7A/B, consists of a MX1CXXC motif where X-1 = T. The same motif, with X-1 = D, is found in metal-binding domains of bacterial zinc transporters, such as ZntA. The Asp is proposed to stabilize divalent over monovalent metals in the binding site, although metal selectivity in vivo appears predominantly governed by protein-protein interactions. To probe the role of T versus D at the X-1 position for Cu transfer in vitro, we created MDCXXC variants of Atox1 and the fourth metal-binding domain of ATP7B, WD4. We find that the mutants bind Cu like the wild-type proteins, but when mixed, in contrast to the wild-type pair, the mutant pair favors Cu-dependent hetero-dimers over directional Cu transport from Atox1 to WD4. Notably, both wild-type and mutant proteins can bind Zn in the absence of competing reducing agents. In presence of zinc, hetero-complexes are strongly favored for both protein pairs. We propose that T is conserved in this motif of Cu-transport proteins to promote directional metal transfer toward ATP7B, without formation of energetic sinks. The ability of both Atox1 and WD4 to bind zinc ions may not be a problem in vivo due to the presence of specific transport chains for Cu and Zn ions.

  • 35.
    Niemiec, Moritz S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weise, Christoph F
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    In vitro thermodynamic dissection of human copper transfer from chaperone to target protein2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 5, p. e36102-Article in journal (Refereed)
    Abstract [en]

    Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

  • 36.
    Nilsson, Lina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nam, Kwangho
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Computational Life Science Center (CLiC), Umeå University,.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Small pH and Salt Variations Radically Alter the Thermal Stability of Metal-Binding Domains in the Copper Transporter, Wilson Disease Protein2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 42, p. 13038-13050Article in journal (Refereed)
    Abstract [en]

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

  • 37. Palm, Maria E
    et al.
    Weise, Christoph F
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundin, Christina
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Wingsle, Gunnar
    Nygren, Yvonne
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Naredi, Peter
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 17, p. 6951-6956Article in journal (Refereed)
    Abstract [en]

    Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

  • 38.
    Palm-Espling, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, David C.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, article id e70473Article in journal (Refereed)
    Abstract [en]

    Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.

  • 39.
    Palm-Espling, Maria E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Role of metal in folding and stability of copper proteins in vitro2012In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1823, no 9, p. 1594-1603Article, review/survey (Refereed)
    Abstract [en]

    Metal coordination is required for function of many proteins. For biosynthesis of proteins coordinating a metal, the question arises if the metal binds before, during or after folding of the polypeptide. Moreover, when the metal is bound to the protein, how does its coordination affect biophysical properties such as stability and dynamics? Understanding how metals are utilized by proteins in cells on a molecular level requires accurate descriptions of the thermodynamic and kinetic parameters involved in protein-metal complexes. Copper is one of the essential transition metals found in the active sites of many key proteins. To avoid toxicity of free copper ions, living systems have developed elaborate copper-transport systems that involve dedicated proteins that facilitate efficient and specific delivery of copper to target proteins. This review describes in vitro and in silico biophysical work assessing the role of copper in folding and stability of copper-binding proteins. Examples of proteins discussed are: a blue-copper protein (Pseudomonas aeruginosa azurin), members of copper-transport systems (bacterial CopZ, human Atox1 and ATP7B domains) and multi-copper ferroxidases (yeast Fet3p and human ceruloplasmin). The consequences of interactions between copper proteins and platinum-complexes are also discussed. 

  • 40.
    Palm-Espling, Maria E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox12012In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 83, no 7, p. 874-881Article in journal (Refereed)
    Abstract [en]

    Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.

  • 41.
    Palm-Espling, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundin, Christina
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Naredi, Peter
    Kirurgi, Sahlgrenska sjukhuset, Göteborgs universitet.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Interaction between anticancer drug Cisplatin and copper chaperone Atox1 in human melanoma cells2014In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 21, no 1, p. 63-68Article in journal (Refereed)
    Abstract [en]

    Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

  • 42. Perdersen, Martin Nors
    et al.
    Fodera, Vito
    Horvath, Istvan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    van Maarschalkerweerd, Andreas
    Toft, Katrine Norgaard
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vestergaard, Bente
    Direct Correlation Between Ligand-Induced alpha-Synuclein Oligomers and Amyloid-like Fibril Growth2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 10422Article in journal (Refereed)
    Abstract [en]

    Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075: protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.

  • 43.
    Petzoldt, Svenja
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Center of Life and Food Sciences, Technische Universität München, Freising, Germany.
    Kahra, Dana
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dingeldein, Artur PG
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Niemiec, Moritz S.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro2015In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, p. 577-585Article in journal (Refereed)
    Abstract [en]

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

  • 44.
    Pozdnyakova, Irina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1804, no 4, p. 740-744Article in journal (Refereed)
    Abstract [en]

    Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.

  • 45. Rodriguez-Granillo, Agustina
    et al.
    Crespo, Alejandro
    Estrin, Dario A
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Copper-transfer mechanism from the human chaperone Atox1 to a metal-binding domain of Wilson disease protein2010In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 10, p. 3698-3706Article in journal (Refereed)
    Abstract [en]

    The molecular details of how copper (Cu) is transferred from the human Cu chaperone Atox1 to metal-binding domains (MBDs) of P(1B)-type ATPases are still unclear. Here, we use a computational approach, employing quantum mechanics/molecular mechanics (QM/MM) methods, to shed light on the reaction mechanism [probable intermediates, Cu(I) coordination geometries, activation barriers, and energetics] of Cu(I) transfer from Atox1 to the fourth MBD of Wilson disease protein (WD4). Both Atox1 and WD4 have solvent-exposed metal-binding motifs with two Cys residues that coordinate Cu(I). After assessing the existence of all possible 2-, 3- and 4-coordinate Cu-intermediate species, one dominant reaction path emerged. First, without activation barrier, WD4's Cys1 binds Cu(I) in Atox1 to form a 3-coordinated intermediate. Next, with an activation barrier of about 9.5 kcal/mol, a second 3-coordinated intermediate forms that involves both of the Cys residues in WD4 and Cys1 of Atox1. This species can then form the product by decoordination of Atox1's Cys1 (barrier of about 8 kcal/mol). Overall, the Cu-transfer reaction from Atox1 to WD4 appears to be kinetically accessible but less energetically favorable (DeltaE = 7.7 kcal/mol). Our results provide unique insights into the molecular mechanism of protein-mediated Cu(I) transfer in the secretory pathway and are in agreement with existing experimental data.

  • 46. Rodriguez-Granillo, Agustina
    et al.
    Crespo, Alejandro
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Conformational dynamics of metal-binding domains in Wilson disease protein: molecular insights into selective copper transfer2009In: Biochemistry, ISSN 1520-4995, Vol. 48, no 25, p. 5849-63Article in journal (Refereed)
    Abstract [en]

    ATP7A/B are human P(1B)-type ATPases involved in cellular Cu homeostasis. The N-terminal parts of these multidomain proteins contain six metal-binding domains (MBDs) connected by linkers. The MBDs are similar in structure to each other and to the human copper chaperone Atox1, although their distinct roles in Cu transfer appear to vary. All domains have the ferredoxin-like fold and a solvent-exposed loop with a MXCXXC motif that can bind Cu(I). Here, we investigated the dynamic behavior of the individual MBDs (WD1-WD6) in ATP7B in apo forms using molecular dynamic simulations. We also performed simulations of three Cu-bound forms (WD2c, WD4c, and WD6c). Our results reveal molecular features that vary distinctly among the MBDs. Whereas WD1, WD2, and WD6 have well-defined Cu loop conformations stabilized by a network of interactions, WD4 and WD5 exhibit greater loop flexibility and, in WD4, helix alpha1 unwinds and rewinds. WD3, which has the lowest sequence identity, behaves differently and its Cu loop is rigid with respect to the rest of the domain. Cu coordination reduces structural dynamics in all domains but WD4c. In agreement with predictions on individual domains, simulations of the six possible Atox1-WD heterocomplexes show that Atox1 interactions with WD4 are the strongest. This study provides molecular explanations for reported Cu transfer and protein-protein interaction specificity.

  • 47. Rodriguez-Granillo, Agustina
    et al.
    Crespo, Alejandro
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Interdomain interactions modulate collective dynamics of the metal-binding domains in the Wilson disease protein2010In: The journal of physical chemistry. B, ISSN 1520-5207, Vol. 114, no 5, p. 1836-48Article in journal (Refereed)
    Abstract [en]

    Wilson disease protein or ATP7B is a key player in human copper (Cu) homeostasis. Belonging to the P(1B) type subfamily of ATPases, its N-terminal region contains six soluble domains (WD1-WD6) connected by linkers that vary in length. These domains share a similar fold and bind Cu(I) in the conserved motif MCXXC. It is unclear why there are six similar domains in the human protein (whereas bacteria and yeast contain only one or two) and why the human metallochaperone Atox1 delivers Cu(I) to only a subset of them. It has been speculated that the extra domains in humans regulate the ATPase in response to different Cu levels, suggesting that, although usually separated by long linkers, the domains can communicate with each other. Here, we performed extensive molecular dynamics simulations on three two-domain constructs in the apo- (WD12, WD34, WD56) and holo- (Cu(I) added to the most C-terminal domain of each construct: WD12c, WD34c and WD56c) forms to investigate how covalent linkage between domains and Cu(I) binding regulate their conformational dynamics. Our results suggest that when linked together the domains do not act as individual units but instead exhibit a distinct pattern of correlated motions, which are domain dependent and modulated by the presence of Cu. Conformational plasticity and degree of reorientation did not correlate with linker length, suggesting strong interdomain communication regardless of the linker length. Our computational findings suggest that cooperativity and long-range communication between domains may be important for the function and regulation of the ATPase.

  • 48. Rodriguez-Granillo, Agustina
    et al.
    Sedlak, Erik
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Stability and ATP Binding of the Nucleotide-binding Domain of the Wilson Disease Protein: Effect of the Common H1069Q Mutation2008In: Journal of Molecular Biology, Vol. 383, no 5, p. 1097-111Article in journal (Refereed)
    Abstract [en]

    Perturbation of the human copper-transporter Wilson disease protein (ATP7B) causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the nucleotide-binding subdomain (N-domain), including the most common mutation, H1069Q. To gain insight into the biophysical behavior of the N-domain under normal and disease conditions, we have characterized wild-type and H1069Q recombinant N-domains in vitro and in silico. We find the mutant to have only 2-fold lower ATP affinity as compared to the wild-type N-domain. Both proteins unfold in an apparent two-state reaction at 20 °C and ATP stabilizes the folded state. The thermal unfolding reactions are irreversible and, for the same scan rate, the wild-type protein is more resistant to perturbation than the mutant. For both proteins, ATP increases the activation barrier towards thermal denaturation. Molecular dynamics simulations identify specific differences in both ATP orientation and protein structure that can explain the absence of catalytic activity for the mutant N-domain. Taken together, our results provide biophysical characteristics that may be general to N-domains in other P1B-ATPases as well as identify changes that may be responsible for the H1069Q WD phenotype in vivo.

  • 49. Rodriguez-Granillo, Agustina
    et al.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Differential Roles of Met10, Thr11, and Lys60 in Structural Dynamics of Human Copper Chaperone Atox12009In: BIOCHEMISTRY including biophysical chemistry & molecular biology, Vol. 48, no 5, p. 960-72Article in journal (Refereed)
    Abstract [en]

    Atox1 is a human copper (Cu) chaperone with the ferredoxin-like fold that binds Cu(I) via two Cys residues in a M10X11C12X13X14C15 motif located in a solvent-exposed loop. Here, we report molecular dynamics simulations that reveal the roles of Met10, Thr11, and Lys60 in Atox1 structural dynamics. Whereas Met10 is conserved in all Atox1 homologues, Thr11 and Lys60 are exchanged for Ser and Tyr in bacteria. From simulations on apo and Cu(I) forms of Met10Ala, Thr11Ala, Lys60Ala, Thr11Ser, and Lys60Tyr variants, we have compared a range of structural and dynamic parameters such as backbone/Cu-loop dynamics, Cys solvent exposure, Cys-Cys distances, and cross-correlated motions. Surprisingly, Atox1 becomes more rigid in the absence of either Thr11 or Lys60, suggesting that these residues introduce protein flexibility. Lys60 and Thr11 also participate in electrostatic networks that stabilize the Cu-bound form and, in the apo form, determine the solvent exposure of the two Cys residues. In contrast, Met10 is buried in the hydrophobic core of Atox1, and its removal results in a dynamic protein structure. Prokaryotic residues are not good substitutes for the eukaryotic counterparts implying early divergence of Cu chaperone homologues. It appears that Atox1 residues have been conserved to ensure backbone/loop flexibility, electrostatic Cu site stabilization, and proper core packing. The discovered built-in flexibility may be directly linked to structural changes needed to form transient Atox1-Cu-target complexes in vivo.

  • 50. Rodriguez-Granillo, Agustina
    et al.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Chemistry.
    Tuning of Copper-Loop Flexibility in Bacillus subtilis CopZ Copper Chaperone: Role of Conserved Residues2009In: The Journal of Physical Chemistry B, Vol. 113, no 7, p. 1919-32Article in journal (Refereed)
    Abstract [en]

    Bacillus subtilis CopZ is a copper (Cu) chaperone that binds and delivers Cu to intracellular targets to maintain cellular Cu homeostasis. Like Cu chaperones from other organisms, including the human homologue Atox1, CopZ has the ferredoxin-like fold and binds Cu(I) via two Cys in a conserved M11X12C13X14X15C16 motif located in a solvent-exposed loop. Here, we have performed extensive molecular dynamics simulations on strategic CopZ variants to reveal structural and dynamic roles of three residues near and in the Cu loop (i.e., Met11, Ser12, and Tyr65). Met11 is conserved in all Cu chaperones, whereas Ser12 and Tyr65 are exchanged for Thr and Lys in eukaryotes like Atox1. Therefore, our simulations included apo and holo forms of Met11Ala, Ser12Ala, and Tyr65Ala, as well as Ser12Thr and Tyr65Lys, CopZ variants. We have discovered that the conserved Met is solvent exposed and important for optimal Cu-loop flexibility in the apo form of CopZ but is buried in the core and aids in packing of the fold in holo-CopZ. Ser12 and Tyr65 are important for assuring Cu-loop flexibility in the apo form; in the Cu-bound form, these residues participate in stabilizing electrostatic networks. The two eukaryotic residues tested are not good substitutes for the prokaryotic counterparts in CopZ. By comparisons to data for Atox1, we conclude that common residues (like Met) and unique residues (like Ser12 and Tyr65 in CopZ) have evolved differentially in prokaryotic and eukaryotic Cu chaperones to tune the flexibility of the Cu loop of the apo form and to provide electrostatic Cu-site stabilization of the holo form.

12 1 - 50 of 69
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf