| 1. |
- Eriksson, Jonas, et al.
(författare)
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Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis
- 2012
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Ingår i: Advances in Yersinia Research. - New York : Springer. - 9781461435600 - 9781461435617 ; , s. 357-363
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Bokkapitel (refereegranskat)abstract
- Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.
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| 2. |
- Hall, Michael, et al.
(författare)
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Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:51, s. 14733-14738
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Tidskriftsartikel (refereegranskat)abstract
- Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
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| 3. |
- Kovermann, Michael, et al.
(författare)
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Structural basis for catalytically restrictive dynamics of a high-energy enzyme state
- 2015
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Ingår i: Nature Communications. - : Macmillan Publishers Ltd.. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible’ states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme’s catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes’ conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.
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| 4. |
- Kovermann, Michael, et al.
(författare)
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Structural basis for ligand binding to an enzyme by a conformational selection pathway
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:24, s. 6298-6303
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-angstrom X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic side-chains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
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| 5. |
- Kulén, Martina, et al.
(författare)
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Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes
- 2018
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 61:9, s. 4165-4175
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein "tunnel" as the main inhibitor binding site (A1), where the compounds participate in an extensive hydrophobic network that restricts the protein's ability to form functional DNA-binding helix−turn−helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-A1 selective PrfA inhibitors with potent antivirulence properties.
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| 6. |
- Lundberg, Erik, et al.
(författare)
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The transthyretin-related protein : structural investigation of a novel protein family
- 2006
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Ingår i: Journal of Structural Biology. - San Diego : Academic Press. - 1047-8477 .- 1095-8657. ; 155:3, s. 445-457
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Tidskriftsartikel (refereegranskat)abstract
- The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.
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| 7. |
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| 8. |
- Verma, Apoorv, et al.
(författare)
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Insights into the evolution of enzymatic specificity and catalysis : from Asgard archaea to human adenylate kinases
- 2022
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 8:44
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.
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| 9. |
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| 10. |
- Addario, Barbara, et al.
(författare)
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Crystallization and preliminary X-ray analysis of the Entamoeba histolytica α-actinin-2 rod domain
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67:10, s. 1214-1217
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Tidskriftsartikel (refereegranskat)abstract
- -Actinins form antiparallel homodimers that are able to cross-link actin filaments. The protein contains three domains: an N-terminal actin-binding domain followed by a central rod domain and a calmodulin-like EF-hand domain at the C-terminus. Here, crystallization of the rod domain of Entamoeba histolytica -actinin-2 is reported; it crystallized in space group P212121, with unit-cell parameters a = 47.8, b = 79.1, c = 141.8 Å. A Matthews coefficient VM of 2.6 Å3 Da-1 suggests that there are two molecules and 52.5% solvent content in the asymmetric unit. A complete native data set extending to a d-spacing of 2.8 Å was collected on beamline I911-2 at MAX-lab, Sweden.
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| 11. |
- Almeida, João R.M., et al.
(författare)
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Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate
- 2023
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Ingår i: Fermentation. - : MDPI AG. - 2311-5637. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Economically feasible bioethanol process from lignocellulose requires efficient fermentation by yeast of all sugars present in the hydrolysate. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth, and fermentation rate, and in addition damage cellular structures. In order to evaluate the capability of S. cerevisiae to adapt and respond to lignocellulosic hydrolysates, the physiological effect of cultivating yeast in the spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome-wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. The adaptation was shown to involve the induction of NADPH-dependent aldehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. These properties were correlated to the expression of several genes encoding oxidoreductases, notably AAD4, ADH6, OYE2/3, and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1, or ATR1. A large set of genes were found to be associated with transcription factors (TFs) involved in stress response (Msn2p, Msn4p, Yap1p) but also cell growth and division (Gcr4p, Ste12p, Sok2p), and these TFs were most likely controlling the response at the post-transcriptional level.
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| 12. |
- Alseekh, Saleh, et al.
(författare)
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Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices
- 2021
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 18:7, s. 747-756
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Forskningsöversikt (refereegranskat)abstract
- This Perspective, from a large group of metabolomics experts, provides best practices and simplified reporting guidelines for practitioners of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics. Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.
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| 13. |
- Altincekic, Nadide, et al.
(författare)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
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Tidskriftsartikel (refereegranskat)abstract
- The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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| 14. |
- Bengtsson, Oskar, et al.
(författare)
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Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.
- 2008
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:11, s. 835-847
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Tidskriftsartikel (refereegranskat)abstract
- Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.
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| 15. |
- Bergdahl, Basti, et al.
(författare)
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Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose
- 2012
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 5:34
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Tidskriftsartikel (refereegranskat)abstract
- Background: The concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production. Results: Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool. Conclusions: The low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.
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| 16. |
- Bian, Xiaolei, et al.
(författare)
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State-of-Health Estimation of Lithium-Ion Batteries by Fusing an Open Circuit Voltage Model and Incremental Capacity Analysis
- 2022
-
Ingår i: IEEE transactions on power electronics. - : Institute of Electrical and Electronics Engineers (IEEE). - 0885-8993 .- 1941-0107. ; 37:2, s. 2226-2236
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Tidskriftsartikel (refereegranskat)abstract
- The state of health (SOH) is a vital parameter enabling the reliability and life diagnostic of lithium-ion batteries. A novel fusion-based SOH estimator is proposed in this study, which combines an open circuit voltage (OCV) model and the incremental capacity analysis. Specifically, a novel OCV model is developed to extract the OCV curve and the associated features-of-interest (FOIs) from the measured terminal voltage during constant-current charge. With the determined OCV model, the disturbance-free incremental capacity (IC) curves can be derived, which enables the extraction of a set of IC morphological FOIs. The extracted model FOI and IC morphological FOIs are further fused for SOH estimation through an artificial neural network. Long-term degradation data obtained from different battery chemistries are used for validation. Results suggest that the proposed fusion-based method manifests itself with high estimation accuracy and high robustness.
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| 17. |
- Blasche, Sonja, et al.
(författare)
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Metabolic cooperation and spatiotemporal niche partitioning in a kefir microbial community
- 2021
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 6:2, s. 196-208
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Tidskriftsartikel (refereegranskat)abstract
- Microbial communities often undergo intricate compositional changes yet also maintain stable coexistence of diverse species. The mechanisms underlying long-term coexistence remain unclear as system-wide studies have been largely limited to engineered communities, ex situ adapted cultures or synthetic assemblies. Here, we show how kefir, a natural milk-fermenting community of prokaryotes (predominantly lactic and acetic acid bacteria) and yeasts (family Saccharomycetaceae), realizes stable coexistence through spatiotemporal orchestration of species and metabolite dynamics. During milk fermentation, kefir grains (a polysaccharide matrix synthesized by kefir microorganisms) grow in mass but remain unchanged in composition. In contrast, the milk is colonized in a sequential manner in which early members open the niche for the followers by making available metabolites such as amino acids and lactate. Through metabolomics, transcriptomics and large-scale mapping of inter-species interactions, we show how microorganisms poorly suited for milk survive in—and even dominate—the community, through metabolic cooperation and uneven partitioning between grain and milk. Overall, our findings reveal how inter-species interactions partitioned in space and time lead to stable coexistence.
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| 18. |
- Bäckström, Stefan, et al.
(författare)
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Crystallization and preliminary studies of the DNA-binding runt domain of AML1.
- 2001
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Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 57:Pt 2, s. 269-71
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Tidskriftsartikel (refereegranskat)abstract
- The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.
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| 19. |
- Bäckström, Stefan, et al.
(författare)
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The RUNX1 Runt domain at 1.25A resolution : a structural switch and specifically bound chloride ions modulate DNA binding.
- 2002
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Ingår i: J Mol Biol. - 0022-2836. ; 322:2, s. 259-72
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Tidskriftsartikel (refereegranskat)abstract
- The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.
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| 20. |
- Canelas, A.B., et al.
(författare)
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Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains
- 2010
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 1:9
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Tidskriftsartikel (refereegranskat)abstract
- The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.
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| 21. |
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| 22. |
- Carlsson, Jonas, 1979-
(författare)
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Mutational effects on protein structure and function
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1).When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations.For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients.The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1.The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements.In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes.A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro.ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated.To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.
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| 23. |
- Colyn Bwanika, Henri, et al.
(författare)
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Limiting the Effects of Radiation Damage in MicroED through Dose Selection during Data Processing
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Microcrystal electron diffraction (MicroED), also known as three-dimensional electron diffraction (3D ED), allows collection of diffraction data from submicron-sized crystals under low electron dose conditions, typically around 5-6 e-Å-2 in total. Despite having several advantages of MicroED over most conventional X-ray crystallographic techniques, susceptibility to radiation damage is a big problem that remains to be solved. Similar to X-ray crystallography, radiation damage to the macromolecular crystal structures in MicroED manifests in two forms, the global damage that affects the overall crystal lattice order and the site-specific damage that affects highly sensitive residues and moieties in macromolecules. In this study, we investigated data processing strategies that could be used to limit the effects of radiation damage to the crystal even when data collection is performed at high electron doses. During MicroED data collection, radiation damage increases with the number of acquired ED frames because the accumulated electron dose increases. To limit the damage, we propose to process only the first few frames of a dataset with a certain low dose cutoff. Data collected from several crystals and processed in this way can be merged to increase completeness and subsequently be used for structure refinement. According to our results, this approach improves the resolution of the data, the data statistics, the structure determination, and the quality of the final structure. The suggested approach could be especially useful in MicroED structure-based drug discovery where atomic resolution structures will provide detailed information about ligand-protein binding properties, which are essential during library screening and hit identification.
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| 24. |
- Dalhus, Bjørn, et al.
(författare)
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Structural basis for thermophilic protein stability : structures of thermophilic and mesophilic malate dehydrogenases.
- 2002
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Ingår i: J Mol Biol. - 0022-2836. ; 318:3, s. 707-21
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Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.
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| 25. |
- Ekström, Fredrik, et al.
(författare)
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Crystallization and X-ray analysis of a bacterial non-haem iron-containing phenylalanine hydroxylase from the Gram-negative opportunistic pathogen Pseudomonas aeruginosa.
- 2003
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Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 59:Pt 7, s. 1310-2
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Tidskriftsartikel (refereegranskat)abstract
- Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment. A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated. The single-domain monomeric protein harbours a non-haem iron at the active site. The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone. Here, the cloning, purification and crystallization of native and SeMet-labelled P. aeruginosa phenylalanine hydroxylase are reported. Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A. Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge.
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| 26. |
- Ekström, Fredrik, 1973-, et al.
(författare)
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Crystallization of the actin-binding domain of human alpha-actinin : analysis of microcrystals of SeMet-labelled protein
- 2003
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Ingår i: Acta Crystallographica Section D. - : Blackwell Munksgaard. - 0907-4449 .- 1399-0047. ; 59:Pt 4, s. 724-726
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Tidskriftsartikel (refereegranskat)abstract
- Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.
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| 27. |
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| 28. |
- Ekström, Fredrik, 1973-
(författare)
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X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylase
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH).To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe.The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum.The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.
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| 29. |
- Fredlund, Elisabeth, et al.
(författare)
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Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala
- 2004
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 70:10, s. 5905-5911
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.
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| 30. |
- Good, James A. D., 1985-, et al.
(författare)
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Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
- 2016
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Ingår i: Cell chemical biology. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 23:3, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 31. |
- Hall, Michael, 1980-, et al.
(författare)
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Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana
- 2012
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 68:3, s. 278-280
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Tidskriftsartikel (refereegranskat)abstract
- The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.
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| 32. |
- Hall, Michael, 1980-, et al.
(författare)
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The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plants
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.
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| 33. |
- Herrgård, Markus J, et al.
(författare)
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A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology
- 2008
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Ingår i: Nature Biotechnology. ; 26:10, s. 1155-1160
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Tidskriftsartikel (refereegranskat)abstract
- Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This make comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChl strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.
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| 34. |
- Hettmer, Simone, et al.
(författare)
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Genetic testing and surveillance in infantile myofibromatosis : a report from the SIOPE Host Genome Working Group
- 2021
-
Ingår i: Familial Cancer. - : Springer. - 1389-9600 .- 1573-7292. ; 20 SI:4, s. 327-336
-
Tidskriftsartikel (refereegranskat)abstract
- Infantile myofibromatosis (IM), which is typically diagnosed in young children, comprises a wide clinical spectrum ranging from inconspicuous solitary soft tissue nodules to multiple disseminated tumors resulting in life-threatening complications. Familial IM follows an autosomal dominant mode of inheritance and is linked toPDGFRBgermline variants. SomaticPDGFRBvariants were also detected in solitary and multifocal IM lesions.PDGFRBvariants associated with IM constitutively activate PDGFRB kinase activity in the absence of its ligand. Germline variants have lower activating capabilities than somatic variants and, thus, require a second cis-acting hit for full receptor activation. Typically, these mutant receptors remain sensitive to tyrosine kinase inhibitors such as imatinib. The SIOPE Host Genome Working Group, consisting of pediatric oncologists, clinical geneticists and scientists, met in January 2020 to discuss recommendations for genetic testing and surveillance for patients who are diagnosed with IM or have a family history of IM/PDGFRBgermline variants. This report provides a brief review of the clinical manifestations and genetics of IM and summarizes our interdisciplinary recommendations.
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| 35. |
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| 36. |
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| 37. |
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| 38. |
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| 39. |
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| 40. |
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| 41. |
- Locmelis, Roland, 1984-
(författare)
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Structural biology studies of thylakoid lumen proteins required for photosystem II assembly and function
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Little is known about the structures and functions of thylakoid lumen proteins. However, some of these proteins have an essential role in photosynthesis. Photosystem II (PSII) complexes are embedded in the thylakoid membrane of oxygenic photosynthetic organisms and one of the central subunits, the D1 protein, is damaged by light during the light driven water – splitting reaction and must be replaced frequently. One of the thylakoid lumen proteins that is essential for assembly and renewal of PSII complexes is the High Chlorophyll Fluorescence 136 (HCF136) protein. Another important protein for the PSII complex assembly is the Low PSII Accumulation Protein 19 (LPA19). Both proteins, HCF136 and LPA19, were shown to bind to the core subunits of the PSII complex from the lumenal side and LPA19 has been shown to explicitly interact with the soluble C-terminus of the D1 protein, one of the core PSII complex proteins. Prior to the replacement of the damaged D1 protein, the PSII complex needs to be disassembled, which is done with the help of the Maintenance of Photosystem II under High light 2 (MPH2) protein. MPH2, also called TL16, is required during the repair cycle of the PSII complex particularly under increased and fluctuating light conditions.In this work I have determined the three-dimensional X-ray structures of the HCF136 protein at 1.6 Å resolution and the LPA19 protein at 1.2 Å resolution and have also biochemically analyzed possible interactions of HCF136 with the C-termini of D1 protein. In addition, we have determined the NMR structure of the MPH2 protein.The protein structures of HCF136, LPA19, and MPH2 determined from A. thaliana provide us with a starting point for further studies to improve our understanding of their functional roles in the assembly, maintenance, disassembly and renewal of the PSII complex. The structures are revealing the molecular details that are particularly important during the design of mutations to study protein-protein interactions and the binding of co-factors.Furthermore, I have contributed to the characterization of AnPrx6, the 1-Cyx peroxiredoxin from Anabaena sp. 7120. Peroxiredoxins are important caretakers of reactive oxygen species and a homolog PrxQ in A.thaliana is found in the thylakoid lumen. The dimeric AnPrx6 protein revealed different active site residues conformations in each of the dimers, which is probably coupled to its enzymatic activity. Unexpectedly, the protein acted also as a chaperone and showed chaperone activity in its dimeric state, which is a novelty for Prx proteins.
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| 42. |
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| 43. |
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| 44. |
- Mishra, Yogesh, et al.
(författare)
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Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7120
- 2017
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C-P) and, if present, the resolving Cys (C-R). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 angstrom resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.
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| 45. |
- Mishra, Yogesh, et al.
(författare)
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Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120
- 2011
-
Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67:10, s. 1203-1206
-
Tidskriftsartikel (refereegranskat)abstract
- Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P212121, with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry1prx) as the template.
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| 46. |
- Nam, Kwangho, et al.
(författare)
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Elucidating dynamics of Adenylate kinase from enzyme opening to ligand release
- 2024
-
Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 64:1, s. 150-163
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Tidskriftsartikel (refereegranskat)abstract
- This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.
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| 47. |
- Ojeda-May, Pedro, et al.
(författare)
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Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions
- 2021
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 60:28, s. 2246-2258
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.
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| 48. |
- Pushkar, Yulia, et al.
(författare)
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Structure and orientation of the Mn4Ca cluster in plant photosystem II membranes studied by polarized range-extended X-ray absorption spectroscopy
- 2007
-
Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 282:10, s. 7198-7208
-
Tidskriftsartikel (refereegranskat)abstract
- X-ray absorption spectroscopy has provided important insights into the structure and function of the Mn4Ca cluster in the oxygen-evolving complex of Photosystem II (PS II). The range of manganese extended x-ray absorption fine structure data collected from PS II until now has been, however, limited by the presence of iron in PS II. Using a crystal spectrometer with high energy resolution to detect solely the manganese K alpha fluorescence, we are able to extend the extended x-ray absorption fine structure range beyond the onset of the iron absorption edge. This results in improvement in resolution of the manganese-backscatterer distances in PS II from 0.14 to 0.09 angstrom. The high resolution data obtained from oriented spinach PS II membranes in the S-1 state show that there are three di-mu-oxo-bridged manganese-manganese distances of similar to 2.7 and similar to 2.8 angstrom in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of similar to 60 degrees relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the similar to 3.2 angstrom manganese-manganese and the similar to 3.4 angstrom manganese-calcium interactions in oriented PS II samples and determine their orientation relative to the membrane normal. The average of the manganese-calcium vectors at similar to 3.4 angstrom is aligned along the membrane normal, while the similar to 3.2 angstrom manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn4Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models.
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| 49. |
- Rogne, Per, et al.
(författare)
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Molecular mechanism of ATP versus GTP selectivity of adenylate kinase
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. 3012-3017
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
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| 50. |
- Schober, Sebastian Johannes, et al.
(författare)
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No Improvement of Survival for Alveolar Rhabdomyosarcoma Patients After HLA-Matched Versus -Mismatched Allogeneic Hematopoietic Stem Cell Transplantation Compared to Standard-of-Care Therapy
- 2022
-
Ingår i: Frontiers in Oncology. - : Frontiers Media S.A.. - 2234-943X. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundPatients with stage IV alveolar rhabdomyosarcoma (RMA) have a 5-year-survival rate not exceeding 30%. Here, we assess the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for these patients in comparison to standard-of-care regimens. We also compare the use of HLA-mismatched vs. HLA-matched grafts after reduced vs. myeloablative conditioning regimens, respectively. Patients and MethodsIn this retrospective analysis, we compare event-free survival (EFS), overall survival (OS), and toxicity of HLA-mismatched vs. -matched transplanted patients in uni- and multivariate analyses (total: n = 50, HLA-matched: n = 15, HLA-mismatched: n = 35). Here, the factors age at diagnosis, age at allo-HSCT, sex, Oberlin score, disease status at allo-HSCT, and HLA graft type are assessed. For 29 primarily transplanted patients, three matched non-transplanted patients per one transplanted patient were identified from the CWS registry. Outcomes were respectively compared for OS and EFS. Matching criteria included sex, age at diagnosis, favorable/unfavorable primary tumor site, and metastatic sites. ResultsMedian EFS and OS did not differ significantly between HLA-mismatched and -matched patients. In the mismatched group, incidence of acute GvHD was 0.87 (grade III-IV: 0.14) vs. 0.80 in HLA-matched patients (grade III-IV: 0.20). Transplant-related mortality (TRM) of all patients was 0.20 and did not differ significantly between HLA-mismatched and -matched groups. A proportion of 0.58 relapsed or progressed and died of disease (HLA-mismatched: 0.66, HLA-matched: 0.53) whereas 0.18 were alive in complete remission (CR) at data collection. Multivariate and competing risk analyses confirmed CR and very good partial response (VGPR) status prior to allo-HSCT as the only decisive predictor for OS (p < 0.001). Matched-pair survival analyses of primarily transplanted patients vs. matched non-transplanted patients also identified disease status prior to allo-HSCT (CR, VGPR) as the only significant predictor for EFS. Here, OS was not affected, however. ConclusionIn this retrospective analysis, only a subgroup of patients with good response at allo-HSCT survived. There was no survival benefit of allo-transplanted patients compared to matched controls, suggesting the absence of a clinically relevant graft-versus-RMA effect in the current setting. The results of this analysis do not support further implementation of allo-HSCT in RMA stage IV patients.
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