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  • 1.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Lindhagen-Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Ca2+ enhances Aβ polymerization rate and fibrillar stability in a dynamic manner2013Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 450, s. 189-197Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Identifying factors that affect the self-assembly of the amyloid-β peptide (Aβ) is of utmost importance in the quest to understand the molecular mechanisms causing Alzheimer's disease (AD). Ca2+ has previously been shown to accelerate both Aβ fibril nucleation and maturation, and a dysregulated Ca2+ homeostasis frequently correlates with development of AD. The mechanisms regarding Ca2+ binding as well as its effect on fibril kinetics are not fully understood. Using a polymerization assay we show that Ca2+ in a dynamic and reversible manner enhances both the elongation rate and fibrillar stability, where specifically the "dock and lock" phase mechanism is enhanced. Through NMR analysis we found that Ca2+ affects the fibrillar architecture. In addition, and unexpectedly, we found that Ca2+ does not bind the free Aβ monomer. This implies that Ca2+ binding requires an architecture adopted by assembled peptides, and consequently is mediated through intermolecular interactions between adjacent peptides. This gives a mechanistic explanation to the enhancing effect on fibril maturation and indicates structural similarities between prefibrillar structures and mature amyloid. Taken together we expose how Ca2+ levels affect the delicate equilibrium between the monomeric and assembled Aβ and how fluctuations in vivo may contribute to development and progression of the disease.

  • 2.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pihl, Mathias
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism2014Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, nr 31, s. 10956-10964Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease is linked to a pathological polymerization of the endogenous amyloid β-peptide (Aβ) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aβ fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (KD), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the KD, polymerization cannot occur. However, the KD for Aβ has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aβ amyloid forms in vivo has been a matter of debate. Using SPR, we found that the KD of Aβ dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aβ to polymerize within a picomolar concentration range that is close to the physiological Aβ concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aβ contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aβ concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

  • 3.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer2011Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, nr 9, s. e25157-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.

  • 4.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brattsand, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Dermatologi och venereologi.
    Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis2012Ingår i: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 393, nr 5, s. 369-377Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.

  • 5. Edman, Maria
    et al.
    Berg, Stefan
    Storm, Patrik
    Wikström, Malin
    Vikström, Susanne
    Öhman, Anders
    Wieslander, Ake
    Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae.2003Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 10Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

  • 6.
    Edwin, Aaron
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Stier, Gunter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae2014Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 96, s. 39-47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

  • 7.
    Edwin, Aaron
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Persson, Cecilia
    Mayzel, Maxim
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Karlsson, B. Göran
    Sauer-Eriksson, A. Elisabeth
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae2015Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, nr 12, s. 2076-2080Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N- terminal domain (residues 23-103) that contains two short alpha-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  • 8.
    Figueira, Joao
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Adolfsson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Psykiatri.
    Nordin Adolfsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Psykiatri.
    Nyberg, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Serum Metabolite Markers of Dementia Through Quantitative NMR Analysis: The Importance of Threonine-Linked Metabolic Pathways2019Ingår i: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 69, nr 3, s. 763-774Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a great need for diagnostic biomarkers of impending dementia. Metabolite markers in blood have been investigated in several studies, but inconclusive findings encourage further investigation, particularly in the pre-diagnostic phase. In the present study, the serum metabolomes of 110 dementia or pre-diagnostic dementia individuals and 201 healthy individuals matched for age, gender, and education were analyzed by nuclear magnetic resonance spectroscopy in combination with multivariate data analysis. 58 metabolites were quantified in each of the 311 samples. Individuals with dementia were discriminated from controls using a panel of seven metabolites, while the pre-diagnostic dementia subjects were distinguished from controls using a separate set of seven metabolites, where threonine was a common significant metabolite in both panels. Metabolite and pathway alterations specific for dementia and pre-diagnostic dementia were identified, in particular a disturbed threonine catabolism at the pre-diagnostic stage that extends to several threonine-linked pathways at the dementia stage.

  • 9.
    Figueira, Joao
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Jonsson, Pär
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Nordin Adolfsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Psykiatri.
    Adolfsson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Psykiatri.
    Nyberg, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper. Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    NMR analysis of the human saliva metabolome distinguishes dementia patients from matched controls2016Ingår i: Molecular Biosystems, ISSN 1742-206X, E-ISSN 1742-2051, Vol. 12, nr 8, s. 2562-2571Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Saliva is a biofluid that is sensitive to metabolic changes and is straightforward to collect in a non-invasive manner, but it is seldom used for metabolite analysis when studying neurodegenerative disorders. We present a procedure for both an untargeted and targeted analysis of the saliva metabolome in which nuclear magnetic resonance (NMR) spectroscopy is used in combination with multivariate data analysis. The applicability of this approach is demonstrated on saliva samples selected from the 25 year prospective Betula study, including samples from dementia subjects with either Alzheimer's disease (AD) or vascular dementia at the time of sampling or who developed it by the next sampling/assessment occasion five years later, and age-, gender-, and education-matched control individuals without dementia. Statistically significant multivariate models were obtained that separated patients with dementia from controls and revealed seven discriminatory metabolites. Dementia patients showed significantly increased concentrations of acetic acid (fold change (fc) = 1.25, p = 2 x 10(-5)), histamine (fc = 1.26, p = 0.019), and propionate (fc = 1.35, p = 0.002), while significantly decreased levels were observed for dimethyl sulfone (fc = 0.81, p = 0.005), glycerol (fc = 0.79, p = 0.04), taurine (fc = 0.70, p = 0.007), and succinate (fc = 0.62, p = 0.008). Histamine, succinate, and taurine are known to be important in AD, and acetic acid and glycerol are involved in related pathways. Dimethyl sulfone and propionate originate from the diet and bacterial flora and might reflect poorer periodontal status in the dementia patients. For these seven metabolites, a weak but statistically significant pre-diagnostic value was observed. Taken together, we present a robust and general NMR analysis approach for studying the saliva metabolome that has potential use for screening and early detection of dementia.

  • 10.
    Figueira, João
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Gouveia-Figueira, Sandra
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Carina
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Lif Holgerson, Pernilla
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Nording, Malin L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated, stimulated, and pure parotid saliva2017Ingår i: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 140, s. 295-300, artikel-id S0731-7085(16)31308-5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Saliva is a readily available biofluid that is sensitive to metabolic changes and can be collected through rapid and non-invasive collection procedures, and it shows great promise for clinical metabolomic studies. This work studied the metabolite composition of, and the differences between, saliva samples collected by unstimulated spitting/drooling, paraffin chewing-stimulated spitting, and parotid gland suction using targeted nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for metabolite quantification. As applied here, these two analytical techniques provide complementary metabolite information and together extend the metabolome coverage with robust NMR quantification of soluble metabolites and sensitive targeted LC-MS/MS analysis of bioactive lipids in specific metabolic pathways. The NMR analysis was performed on ultrafiltrated (3kDa cutoff) saliva samples and resulted in a total of 45 quantified metabolites. The LC-MS/MS analysis was performed on both filtered and unfiltered samples and resulted in the quantification of two endocannabinoids (AEA and PEA) and 22 oxylipins, which at present is the most comprehensive targeted analysis of bioactive lipids in human saliva. Important differences in the metabolite composition were observed between the three saliva sample collection methods, which should be taken into consideration when designing metabolomic studies of saliva. Furthermore, the combined use of the two metabolomics platforms (NMR and LC-MS/MS) proved to be viable for research and clinical studies of the salivary metabolome.

  • 11. Ge, Changrong
    et al.
    Georgiev, Alexander
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Wieslander, Åke
    Kelly, Amelie A.
    Tryptophan Residues Promote Membrane Association for a Plant Lipid Glycosyltransferase Involved in Phosphate Stress2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 8, s. 6669-6684Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

  • 12. Haglund, Ellinor
    et al.
    Lind, Jesper
    Öman, Tommy
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Mäler, Lena
    Oliveberg, Mikael
    The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway2009Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, nr 51, s. 21619-21624Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

  • 13.
    Jia, Xueen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Liu, Yonggang
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Morozova-Roche, Ludmilla A
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 414, nr 5, s. 699-712Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.

  • 14. Lycksell, P O
    et al.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Bengtsson-Olivecrona, G
    Johansson, L B
    Wijmenga, S S
    Wernic, D
    Gräslund, A
    Sequence specific 1H-NMR assignments and secondary structure of a carboxy-terminal functional fragment of apolipoprotein CII.1992Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 205, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The structural properties of a synthetic fragment of human apolipoprotein CII (apoCII) has been studied by circular dichroism and proton nuclear magnetic resonance. The fragment corresponds to the carboxy-terminal 30 amino acid residues and retains the ability of apoCII to activate lipoprotein lipase. Like native apoCII, the fragment has a tendency to self-associate in pure aqueous solution. Addition of 1,1,1,3,3,3-hexafluoro-2-isopropanol to aqueous solvent dissolves the aggregates and leads to an increase in the alpha-helical content of the peptide, probably by stabilizing transient helical structures. The resonances in the 1H-NMR spectrum of the fragment in 35% (CF3)2CHOH were assigned through standard procedures from nuclear Overhauser enhancement spectroscopy, correlated spectroscopy and total correlated spectroscopy experiments. The NMR data indicates the formation of a stable alpha helix spanning Ile66-Gly77. Another alpha helical turn may be formed between Lys55 and Ala59 and possibly span even further towards the carboxyl terminus. These structural elements are different from those previously predicted for this part of the sequence of apoCII.

  • 15.
    Morozova-Roche, Ludmilla
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Zamotin, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Malisauskas, Mantas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chertkova, Rita
    Lavrikova, Marika A
    Kostanyan, Irina A
    Dolgikh, Dmiltry A
    Kirpichnikov, Michail P
    Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways2004Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Biochemistry, Vol. 43, nr 30, s. 9610-9619Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-2 octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and -sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

  • 16.
    Olofsson, Anders
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Ippel, Johannes H
    Wijmenga, Sybren S
    Lundgren, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ohman, Anders
    Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy.2004Ingår i: J Biol Chem, ISSN 0021-9258, Vol. 279, nr 7, s. 5699-707Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.

  • 17.
    Olofsson, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindhagen Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vestling, Monika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Quenched hydrogen/deuterium exchange NMR characterization of amyloid-β peptide aggregates formed in the presence of Cu2+ or Zn2+2009Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, nr 15, s. 4051-4060Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease, a neurodegenerative disorder causing synaptic impairment and neuronal cell death, is strongly correlated with aggregation of the amyloid-β peptide (Aβ). Divalent metal ions such as Cu2+ and Zn2+ are known to significantly affect the rate of aggregation and morphology of Aβ assemblies in vitro and are also found at elevated levels within cerebral plaques in vivo. The present investigation characterized the architecture of the aggregated forms of Aβ(1–40) and Aβ(1–42) in the presence or absence of either Cu2+ or Zn2+ using quenched hydrogen/deuterium exchange combined with solution NMR spectroscopy. The NMR analyses provide a quantitative and residue-specific structural characterization of metal-induced Aβ aggregates, showing that both the peptide sequence and the type of metal ion exert an impact on the final architecture. Common features among the metal-complexed peptide aggregates are two solvent-protected regions with an intervening minimum centered at Asn27, and a solvent-accessible N-terminal region, Asp1–Lys16. Our results suggest that Aβ in complex with either Cu2+ or Zn2+ can attain an aggregation-prone β-strand–turn–β-strand motif, similar to the motif found in fibrils, but where the metal binding to the N-terminal region guides the peptide into an assembly distinctly different from the fibril form.

  • 18.
    Olofsson, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Lindhagen-Persson, Malin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Amide solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloid-beta(1-42) form different fibrillar structures under identical conditions.2007Ingår i: Biochem J, ISSN 1470-8728, Vol. 404, nr 1, s. 63-70Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39–43 residue long Ab (amyloid-b)-peptide. The most abundant species, Ab(1–40) and Ab(1–42), are both present within senile plaques, but Ab(1–42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Ab-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Ab(1–40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Ab(1–42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Ab(1–40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two b-strands connected with a bend. This protection pattern partly resembles the pattern found in Ab(1–42) fibrils, but the Ab(1–40) fibrils display a significantly increased protection for the N-terminal residues Phe4–His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37–Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.

  • 19.
    Olofsson, Anders
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP).
    Amyloid fibril dynamics revealed by combined hydrogen/deuterium exchange and nuclear magnetic resonance2009Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 385, nr 2, s. 374-376Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A general method to explore the dynamic nature of amyloid fibrils is described, combining hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy to determine the exchange rates of individual amide protons within an amyloid fibril. Our method was applied to fibrils formed by the amyloid-beta(1-40) peptide, the major protein component of amyloid plaques in Alzheimer's disease. The fastest exchange rates were detected among the first 14 residues of the peptide, a stretch known to be poorly structured within the fibril. Considerably slower exchange rates were observed in the remainder of the peptide within the beta-strand-turn-beta-strand motif that constitutes the fibrillar core.

  • 20.
    Olofsson, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    The solvent protection of alzheimer amyloid-beta-(1-42) fibrils as determined by solution NMR spectroscopy.2006Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 281, nr 1, s. 477-83Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer disease is a neurodegenerative disorder that is tightly linked to the self-assembly and amyloid formation of the 39-43-residue-long amyloid-beta (Abeta) peptide. Considerable evidence suggests a correlation between Alzheimer disease development and the longer variants of the peptide, Abeta-(1-42/43). Currently, a molecular understanding for this behavior is lacking. In the present study, we have investigated the hydrogen/deuterium exchange of Abeta-(1-42) fibrils under physiological conditions, using solution NMR spectroscopy. The obtained residue-specific and quantitative map of the solvent protection within the Abeta-(1-42) fibril shows that there are two protected core regions, Glu11-Gly25 and Lys28-Ala42, and that the residues in between, Ser26 and Asn27, as well as those in the N terminus, Asp1-Tyr10, are solvent-accessible. This result reveals considerable discrepancies when compared with a previous investigation on Abeta-(1-40) fibrils and suggests that the additional residues in Abeta-(1-42), Ile41 and Ala42, significantly increase the solvent protection and stability of the C-terminal region Lys28-Ala42. Consequently, our findings provide a molecular explanation for the increased amyloidogenicity and toxicity of Abeta-(1-42) compared with shorter Abeta variants found in vivo.

  • 21.
    Paracuellos, Patricia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sauer-Eriksson, A Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Expression and purification of SfaX(II), a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli2012Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 86, nr 2, s. 127-134Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pathogenic Escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfa(II) fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaX(II) gene, located at the distal end of the sfa(II) operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic E. coli (ExPEC). Until now, detailed characterization of the SfaX(II) protein has been hampered by difficulties in obtaining large quantities of soluble protein. By a rational modeling approach, we engineered a Cys70Ser mutation, which successfully improved solubility of the protein. Here, we present the expression, purification, and initial characterization of the recombinant SfaX(IIC70S) mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaX(IIC70S) protein N-terminally fused with a six histidine (H6) sequence followed by a ZZ tag (a derivative of the Staphylococcus protein A) (H6-ZZ tag). The H6-ZZ tag was cleaved off with Tobacco Etch Virus (TEV) protease and the 166 amino acid full-length homo-dimeric protein was purified using affinity and size-exclusion chromatography. Electrophoretic mobility gel shift assays and atomic force microscopy demonstrated that the protein possesses DNA-binding properties, suggesting that the transcriptional regulatory activity of SfaX(II) can be mediated via direct binding to DNA.

  • 22.
    Petzold, Katja
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Backman, Lars
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Folding of the αΙΙ-spectrin SH3 domain under physiological salt conditions2008Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 474, nr 1, s. 39-47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of II-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the II-spectrin SH3 domain.

  • 23. Wennerberg, Anders B.A.
    et al.
    Jackson, Michael
    Öhman, Anders
    Gräslund, Astrid
    Langel, Ülo
    Bartfai, Tamas
    Rigler, Rudolf
    Mantsch, Henry H.
    The structure of the rodent and porcine neuropeptide galanin and antagonists as determined by FTIR and CD spectroscopy1994Ingår i: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 72, nr 6, s. 1495-1499Artikel i tidskrift (Refereegranskat)
  • 24.
    Wikström Hultdin, Ulrika
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Stina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Allgardsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Stier, Günter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, nr Pt 3, s. 337-341Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.

  • 25.
    Wu, Junfang
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Domellöf, Magnus
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Pediatrik.
    Zivkovic, Angela M.
    Larsson, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Nording, Malin L.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation2016Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 469, nr 3, s. 626-632Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol water (MeOH/H2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9-24 days after delivery) and late (31-87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant.

  • 26.
    Wu, Junfang
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Wuolikainen, Anna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Trupp, Miles
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Jonsson, Pär
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Marklund, Stefan L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Klinisk kemi.
    Andersen, Peter M.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Forsgren, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    NMR analysis of the CSF and plasma metabolome of rigorously matched amyotrophic lateral sclerosis, Parkinson's disease and control subjects2016Ingår i: Metabolomics, ISSN 1573-3882, E-ISSN 1573-3890, Vol. 12, nr 6, artikel-id 101Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Introduction: Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomarkers are absent.

    Objectives: To identify metabolite biomarkers for ALS and PD, and to gain insights into which metabolic pathways are involved in disease.

    Methods: Nuclear magnetic resonance (NMR) metabolomics was utilized to characterize the metabolite profiles of cerebrospinal fluid (CSF) and plasma from individuals in three age, gender, and sampling-date matched groups, comprising 22 ALS, 22 PD and 28 control subjects.

    Results: Multivariate analysis of NMR data generated robust discriminatory models for separation of ALS from control subjects. ALS patients showed increased concentrations of several metabolites in both CSF and plasma, these are alanine (CSF fold change = 1.22, p = 0.005), creatine (CSF-fc = 1.17, p = 0.001), glucose (CSF-fc = 1.11, p = 0.036), isoleucine (CSF-fc = 1.24, p = 0.002), and valine (CSF-fc = 1.17, p = 0.014). Additional metabolites in CSF (creatinine, dimethylamine and lactic acid) and plasma (acetic acid, glutamic acid, histidine, leucine, pyruvate and tyrosine) were also important for this discrimination. Similarly, panels of CSF-metabolites that discriminate PD from ALS and control subjects were identified.

    Conclusions: The results for the ALS patients suggest an affected creatine/creatinine pathway and an altered branched chain amino acid (BCAA) metabolism, and suggest links to glucose and energy metabolism. Putative metabolic markers specific for ALS (e.g. creatinine and lactic acid) and PD (e.g. 3-hydroxyisovaleric acid and mannose) were identified, while several (e.g. creatine and BCAAs) were shared between ALS and PD, suggesting some overlap in metabolic alterations in these disorders.

  • 27. Öhman, Anders
    et al.
    Davydov, R
    Backlund, B M
    Langel, U
    Gräslund, A
    A study of melittin, motilin and galanin in reversed micellar environments, using circular dichroism spectroscopy.1996Ingår i: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 59, nr 1-2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Circular dichroism spectroscopy has been used to study the behaviour of the cytolytic peptide melittin, the intestinal peptide hormone motilin (porcine) and the neuropeptide galanin (porcine) in various reversed micellar systems. The micellar systems used contained sodium dodecyl sulphate, bis(2-ethylhexyl) sulfosuccinate, n-dodecyltrimethylammonium chloride or polyoxyethylene(7) lauryl ether. Various structural changes of the peptides, induced either by varying the water content or the surface charge of the reversed micelles, could be monitored. Melittin has in all micellar systems a large amount of alpha-helix, and is almost unaffected by both water content and the surface charge of the reversed micelles. Motilin on the other hand attains an alpha-helical structure at low water content only. The surface charges seem to be of importance for the association between motilin and the hydrated reversed micellar surface. Galanin has the most complicated behaviour with a large dependence on surface charge and with a water content dependence which varies with the surfactant used. Stabilization of alpha-helical secondary structures was only seen in negatively charged reversed micelles. These observations indicate a specific interaction between galanin and surfactant, probably of electrostatic nature.

  • 28.
    Öhman, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Forsgren, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    NMR metabonomics of cerebrospinal fluid distinguishes between Parkinson's disease and controls2015Ingår i: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 594, s. 36-39Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This study assesses if nuclear magnetic resonance (NMR) metabonomics can discriminate between Parkinson's disease (PD) patients and control subjects, and consequently identify metabolic markers for the disease. One-dimensional H-1 NMR spectroscopy was used for quantitative analysis of metabolites in the cerebrospinal fluid (CSF) from 10 PD patients and 10 control individuals, together with uni- and multivariate statistical analysis to discriminate between the groups and to identify significantly altered metabolite concentrations. In total 60 metabolites were identified and of those 38 were quantified in all CSF samples. An overall lowering of metabolite content was observed in PD patients compared to control subjects (fold change of 0.85 +/- 0.30). Multivariate statistics reveal significant changes (vertical bar w*vertical bar>0.2) among nine metabolites (alanine, creatinine, dimethylamine, glucose, lactate, mannose, phenylalanine, 3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid). Three of these (alanine, creatinine and mannose) are identified as significantly changed also by univariate statistics (p < 0.00132, Bonferroni corrected). Panels with all or a selected set of these metabolites were successfully used for discriminating between the two groups. In conclusion, NMR metabonomics can readily determine metabolite concentrations in CSF, identify putative biomarkers that distinguish between the PD patients and control subjects, and thus potentially become a tool for diagnostic purposes.

  • 29. Öhman, Anders
    et al.
    Lycksell, P O
    Andell, S
    Langel, U
    Bartfai, T
    Gräslund, A
    Solvent stabilized solution structures of galanin and galanin analogs, studied by circular dichroism spectroscopy.1995Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1236, nr 2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Circular dichroism spectroscopy has been used to study how different solvents stabilize secondary structure in the neuropeptide galanin (rat), two N-terminal fragments of galanin, galanin(1-12) and galanin(1-16), and six other differently charged analogs. Among these analogs, the peptide M40, galanin(1-13)-Pro-Pro-Ala-Leu-Ala-Leu-Ala amide, is a high affinity, receptor subtype specific galanin receptor antagonist. The different solvents include sodium dodecyl sulfate (SDS) micelle solutions, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) vesicle solutions. 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and 100% 2,2,2-trifluoroethanol (TFE). DOPC vesicles did not change the structure of the peptides as compared to aqueous solvent. The negatively charged DOPG vesicles and SDS micelles induced similar changes towards alpha-helical structures in all peptides. The HFP and TFE solvents have an even stronger tendency to stabilize alpha-helical conformations in these peptides. Since DOPG vesicles can be considered as a model system for negatively charged biological membranes, the solution structures observed in the presence of DOPG or SDS may be the most relevant for the in vivo situation. Correlations between the binding affinity of the peptides to hippocampal galanin receptors and their observed structures in the DOPG solvent were investigated.

  • 30.
    Öhman, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Lycksell, P O
    Gräslund, A
    A refined three-dimensional solution structure of a carboxy terminal fragment of apolipoprotein CII.1993Ingår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 22, nr 5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The three-dimensional structure of a synthetic fragment of human apolipoprotein CII (apo-CII) in 35%, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) has been determined on the basis of distance and intensity constraints derived from two-dimensional proton nuclear magnetic resonance measurements. The NOE crosspeak build-up rates were converted to distance constraints which were used in the distance geometry program DI-ANA. A set of one hundred structures were generated and of these ten structures were used in molecular dynamics simulations using the program XPLOR. This program enabled a direct minimization between the difference of the two-dimensional NOE intensities and those calculated from the full relaxation matrix. In this way spin diffusion is fully taken into account, which can be seen from the considerable improvement of the R-factor after the relaxation matrix refinement. These calculations show that this fragment, which corresponds to the carboxy terminal 30 amino acids of intact apo-CII and which retains its ability to activate lipoprotein lipase, is essentially flexible, but has three defined secondary structural elements. The most significant one is an alpha-helix between residues 67 and 74. The following three residues adopt a turn-like structure. Another turn of alpha-helix is seen between residues 56 and 59. The effect of the solvent system on the secondary structure was studied by circular dichroism spectroscopy. The results show that the mixed aqueous 35% HFP solvent induces secondary structure of a very similar nature to the one induced by sodium dodecyl sulphate.

  • 31. Öhman, Anders
    et al.
    Lycksell, P O
    Juréus, A
    Langel, U
    Bartfai, T
    Gräslund, A
    NMR study of the conformation and localization of porcine galanin in SDS micelles. Comparison with an inactive analog and a galanin receptor antagonist.1998Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 37, nr 25Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Galanin is a 29/30-residue neuro-endocrine peptide which performs its many important physiological functions via a membrane-bound receptor. By using two-dimensional proton NMR spectroscopy, complete relaxation matrix analysis, and simulated annealing, the conformation of porcine galanin was determined in a membrane-mimicking solvent containing sodium dodecyl sulfate (SDS) micelles. The final family of calculated structures displays three well-defined beta- or gamma-turn regions, comprising residues 1-5, 7-10, and 24-27, but has otherwise a random conformation. The receptor-interacting N-terminal part, residues 1-5, was found to be best defined with a backbone RMSD value of 0.12 A. The mode of association between galanin and the SDS micelle was determined by observing the broadening effect on proton resonances, when spin-labeled 5- and 12-doxyl stearate molecules were added. It was concluded that galanin is located close to the surface of the micelle with two regions, residues 6-9 and 24-29, as well as two single residues, 18 and 21, reaching out into the aqueous solvent. Additional NMR studies were carried out on an inactive analogue, Ala2-galanin, and an antagonist M40. The results show that the proton resonances of galanin and M40 have identical chemical shifts in the N-terminal receptor-interacting region, indicating similar solution structures in this region. For Ala2-galanin, the same region displays a spectral heterogeneity with chemical shifts clearly different from the other two peptides, indicative of different secondary structures. These results may provide a structural background for the antagonist activity of M40 and the hormonal inactivity of Ala2-galanin, as compared to galanin.

  • 32.
    Öhman, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Rak, Alexey
    Dontsova, Maria
    Garber, Maria B
    Härd, Torleif
    NMR structure of the ribosomal protein L23 from Thermus thermophilus.2003Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 26, nr 2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.

  • 33.
    Öhman, Anders
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Öman, Tommy
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Oliveberg, Mikael
    Solution structures and backbone dynamics of the ribosomal protein S6 and its permutant P(54-55)2010Ingår i: Protein science : a publication of the Protein Society, ISSN 1469-896X, Vol. 19, nr 1, s. 183-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96-residue P(54-55), and the structure of its 101-residue parent protein S6(wt) in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6(wt), the overall solution structures of both P(54-55) and S6(wt) show a beta-sheet of four anti-parallel beta-strands with two alpha-helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6(wt) reveals a disordered loop in the region between beta-strands 2 and 3 (Leu43-Phe60) instead of a well-ordered stretch and associated hydrophobic mini-core observed in the crystal structure. Moreover, the data for P(54-55) show that the joined wild-type N- and C-terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6(wt) structure is relatively insensitive to mutational perturbations, and why P(54-55) is more stable than S6(wt): the permutant incision at Lys54-Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild-type N- and C-termini is stabilizing.

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