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  • 1.
    Abelein, Axel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Modulation of Alzheimer's amyloid β peptide self-assembly: Insights into molecular mechanisms of peptide aggregation associated with Alzheimer's disease2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Misfolding of proteins and peptides is closely linked to several neurodegenerative disorders, among them Alzheimer's disease (AD), the most prominent example of brain diseases. The self-assembly of the amyloid β peptide (Aβ) into amyloid fibrils is one histologic hallmark of AD. A detailed knowledge about the underlying mechanism(s) of Aβ aggregation is crucial for advances toward a fundamental understanding of the disease, which may promote the search for and design of efficient therapeutics. The work presented in this thesis deals with modulation of the aggregation process by various compounds, i.e. small organic molecules (e.g. lacmoid and Congo red), surfactants and metal ions. These results provide insight into the molecular mechanism of modulator interactions and interference with Aβ and its aggregation pathways. Applying a combination of kinetic and dynamic studies as well as structural investigations we characterized the molecular interactions between Aβ and aggregation modulators in terms of microscopic rate constants, conformational preferences and thermodynamics. An important conclusion is that these modulators form highly dynamic complexes with Aβ, with life-times on the timescale of milliseconds. Despite the similar exchange dynamics, the effect on peptide aggregation is modulator-specific and fibril formation can be accelerated, retarded or inhibited by their interactions. In summary, Aβ self-assembly is governed by microscopic kinetic and dynamic processes that can be altered by aggregation modulators. Further elucidation of these mechanisms is beneficial for the understanding and therapeutic intervention of amyloid diseases.

  • 2.
    Abelein, Axel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Modulation of amyloid β peptide self-assembly: Aggregation mechanisms associated with Alzheimer's disease2013Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
  • 3.
    Abelein, Axel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The zinc ion – a minimal chaperone mimicking agent forretardation of amyloid β peptide fibril formationManuskript (preprint) (Övrigt vetenskapligt)
  • 4.
    Abelein, Axel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lang, Lisa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lendel, Christofer
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Corrigendum to “Transient small molecule interactions kinetically modulate amyloid β peptide self-assembly” [FEBS Lett. 586 (2012) 3991–3995]2013Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, nr 9, s. 1452-1452Artikel i tidskrift (Övrigt vetenskapligt)
  • 5. Ahn, Young O.
    et al.
    Mahinthichaichan, Paween
    Lee, Hyun Ju
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ouyang, Hanlin
    Kaluka, Daniel
    Yeh, Syun-Ru
    Arjona, Davinia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rousseau, Denis L.
    Tajkhorshid, Emad
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    Conformational coupling between the active site and residues within the K-C-channel of the Vibrio cholerae cbb(3)-type (C-family) oxygen reductase2014Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, nr 42, s. E4419-E4428Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K-C-channel is a conserved glutamate in subunit III. However, the majority of the K-C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K-C-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb(3) eliminate catalytic activity, but also cause perturbations that propagate over a 28-angstrom distance to the active site heme b(3). The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix alpha 7, which contains both Y241(Vc) and the active site crosslinked Y255(Vc), as well as two Cu-B histidine ligands. Other mutations of residues within or near helix alpha 7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

  • 6.
    Alikhani, Nyosha
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berglund, Anna-Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Engmann, Tanja
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Voegtle, F. -Nora
    Pavlov, Pavel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Meisinger, Chris
    Langer, Thomas
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species2011Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 410, nr 3, s. 400-410Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.

  • 7.
    Allard, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Nuclear magnetic resonance studies of nucleotide-protein interactions and of peptide structure-dynamics relations1994Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 8.
    Andersson, August
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Application of isotropic bicelles as model membranes2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear 1H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration.

    NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects.

    Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.

  • 9.
    Andersson, Charlotta S.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berthold, Catrine L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A dynamic c terminal segment in the mycobacterium tuberculosis mn/fe r2lox protein can adopt a helical structure with possible functional consequences2012Ingår i: Chemistry and Biodiversity, ISSN 1612-1872, E-ISSN 1612-1880, Vol. 9, nr 9, s. 1981-1988Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.

  • 10. Arukuusk, Piret
    et al.
    Paernaste, Ly
    Oskolkov, Nikita
    Copolovici, Dana-Maria
    Margus, Helerin
    Padari, Kaert
    Moell, Kaidi
    Maslovskaja, Julia
    Tegova, Radi
    Kivi, Gaily
    Tover, Andres
    Pooga, Margus
    Ustav, Mart
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids2013Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, nr 5, s. 1365-1373Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

  • 11.
    Arvidsson, Klas G. J.
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Solution structure and dynamics of peptides as studied by NMR and CD: a search for correlations between structure and biological function1994Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 12.
    Bajinskis, Ainars
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Lindegren, Helene
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Lotta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Harms-Ringdahl, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells2011Ingår i: Radiation Research, ISSN 0033-7587, E-ISSN 1938-5404, Vol. 175, nr 2, s. 185-192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose gamma-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs gamma rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate gamma rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism. (C) 2011 by Radiation Research Society

  • 13. Bartelink, Eric J.
    et al.
    Sholts, Sabrina B.
    Milligan, Colleen F.
    Van Deest, Traci L.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Linköping University, Sweden.
    A Case of Contested Cremains Analyzed Through Metric and Chemical Comparison2015Ingår i: Journal of Forensic Sciences, ISSN 0022-1198, E-ISSN 1556-4029, Vol. 60, nr 4, s. 1068-1073Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Since the 1980s, cremation has become the fastest growing area of the U.S. funeral industry. At the same time, the number of litigations against funeral homes and cremation facilities has increased. Forensic anthropologists are often asked to determine whether the contents of an urn are actually cremated bone, and to address questions regarding the identity of the remains. This study uses both metric and chemical analyses for resolving a case of contested cremains. A cremains weight of 2021.8 g was predicted based on the decedent's reported stature and weight. However, the urn contents weighed 4173.5 g. The urn contents also contained material inconsistent with cremains (e.g., moist sediment, stones, ferrous metal). Analysis using XRD and SEM demonstrated that the urn contained thermally altered bone as well as inorganic material consistent with glass fiber cement. Although forensically challenging, cremains cases such as this one can be resolved using a multidisciplinary approach.

  • 14.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structure and Dynamics of Membrane Associated Peptides2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The peptide-membrane interaction is a key element for many biological functions, from cell signaling to cell internalization. In this thesis the peptide-membrane interaction of six different peptides have been studied with respect to their structure, membrane location and dynamics with spectroscopic methods. Penetratin and the N-terminal sequence of the bovine prion protein (1-30), bPrPp, belong to a class of peptides called cell-penetrating peptides (CPPs). CPPs are short, often highly basic peptides that have the ability to facilitate translocation of an attached hydrophilic cargo over cell-membrane. CD and NMR spectroscopy reveled that penetratin, the (supposedly) non-penetrating mutant pentratin(W48F,W56F) and bPrPp are all highly helical in membrane mimicking media. The position with respect to the bilayer is, however, very different for the three peptides, Penetratin is residing on the membrane surface with a slight tilt while bPrPp is transmembrane and penetratin(W48F,W56F) is somewere in between. These differences can explain the different impact these peptides have on membranes

    We have also shown that penetratin can escape from vesicles when an electrochemical or pH gradient is present over the membrane, which support endocytotic internalization.

    Melittin is a 26 amino acid long residue long peptide and is the major component of the European honey bee venom. Many studies have shown that melittin induces a transient pore that causes leakage in both natural and artificial membranes. In paper IV we used melittin as a model-peptide to investigate how peptides affect lipid dynamics in model-membranes. We showed that carbon-13 relaxation of the lipids could be used to characterize peptide induced changes in lipid dynamics

    The voltage sensor is a domain of the voltage-dependent potassium channel containing several positively charged amino acids (usually arginines). The sensor undergoes a conformational change as a response to a membrane potential. Here, we have studied the membrane location of two fragments corresponding to the “paddle” domain of two different potassium channels, KvAP and HsapBK. NMR and fluorescence studies indicate that both peptides reside inside of the hydrophobic interior of the bilayer, which show that the fragment behave the same way as it does in the intact protein.

    All six of these peptides interact strongly with model-membranes and adopt a helical conformation even though they have very different biological function. The difference in biological function can instead be explained by the variation in membrane position and membrane dynamics of these peptides

  • 15.
    Björnerås, Johannes
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dynorphin A – Interactions with receptors and the membrane bilayer2013Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The work presented in this thesis concerns the dynorphin neuropeptides, and dynorphin A (DynA) in particular. DynA belongs to the wider class of typical opioid peptides that, together with the opioid receptors, a four-membered family of GPCR membrane proteins, form the opioid system. This biological system is involved or implicated in several physiological processes such as analgesia, addiction and depression, and effects caused by DynA through this system, mainly through interaction with the kappa subtype of the opioid receptors (KOR), are called the opioid effects. In addition to this, non-opioid routes of action for DynA have been proposed, and earlier studies have shown that direct membrane interaction is likely to contribute to these non-opioid effects. The results discussed here fall into either of two categories; the interaction between DynA and a fragment of KOR, and the direct lipid interaction of DynA and two variant peptides.For the receptor interaction case, DynA most likely causes its physiological effects through binding its N-terminal into a transmembrane site of the receptor protein, while the extracellular regions of the protein, in particular the extracellular loop II (EL2), have been shown to be important for modulating the selectivity of KOR for DynA. Here we have focussed on the EL2, and show the feasibility of transferring this sequence into a soluble protein scaffold. Studies, predominantly by nuclear magnetic resonance (NMR) spectroscopy, of EL2 in this new environment show that the segment has the conformational freedom expected of a disordered loop sequence, while the scaffold keeps its native beta-barrel fold. NMR chemical shift and paramagnetic resonance enhancement experiments show that DynA binds with high specificity to EL2 with a dissociation constant of approximately 30 micro Molar, while binding to the free EL2 peptide is an order of magnitude weaker. The strength of these interactions are reasonable for a receptor recognition event. No binding to the naked scaffold protein is observed.In the second project, the molecules of interest were two DynA peptide variants recently found in humans and linked to a neurological disorder. Previously published reports from our group and collaborators pointed at very different membrane-perturbing properties for the two variants, and here we present the results of a follow-up study, where the variants R6W-DynA and L5S-DynA were studied by NMR and circular dichroism (CD) spectroscopy in solutions of fast-tumbling phospholipid bicelles, and compared with wild type DynA. Our results show that R6W-DynA interacts slightly stronger with lipids compared to wild type DynA, and much stronger compared to L5S-DynA, in terms of bicelle association, penetration and structure induction. These results are helpful for explaining the differences in toxicity, membrane perturbation and relationship to disease, between the studied neuropeptides.

  • 16.
    Björnerås, Johannes
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The opioid peptide dynorphin A: Biophysical studies of peptide–receptor and peptide–membrane interactions2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA.

    The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event.

    With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge.

    Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.

  • 17.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Botana, Adolfo
    Morris, Gareth A.
    Nilsson, Mathias
    Resolving complex mixtures: trilinear diffusion data2014Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 58, nr 4, s. 251-257Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Complex mixtures are at the heart of biology, and biomacromolecules almost always exhibit their function in a mixture, e.g., the mode of action for a spider venom is typically dependent on a cocktail of compounds, not just the protein. Information about diseases is encoded in body fluids such as urine and plasma in the form of metabolite concentrations determined by the actions of enzymes. To understand better what is happening in real living systems we urgently need better methods to characterize such mixtures. In this paper we describe a potent way to disentangle the NMR spectra of mixture components, by exploiting data that vary independently in three or more dimensions, allowing the use of powerful algorithms to decompose the data to extract the information sought. The particular focus of this paper is on NMR diffusion data, which are typically bilinear but can be extended by a third dimension to give the desired data structure.

  • 18.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Interaction of Disease-Related Dynorphin A Variants2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 24, s. 4157-4167Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small a-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (similar to 90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (similar to 50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

  • 19.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The membrane interaction of dynorphin A depends on lipid head-group chargeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The influence of lipid bicelles on the dynamics of the opioid peptide DynA has been investigated by Nuclear Magnetic Resonance. DynA exerts its opioid effects mainly through interactions with the κ subtype of the opioid receptors, but has also been demonstrated to have direct interactions with membranes. Among other properties, it has been shown that the peptide causes membrane disruption and may penetrate bilayers. Despite the fact that DynA appears to bind tightly to model lipid bilayers, no structure induction has been observed. To further study the effect of membrane interactions we have here therefore measured the fast local dynamics of DynA specifically labeled with 15N in three backbone amide sites (Gly2, Leu5 and Leu12) in fast-tumbling bicelles, both with and without the incorporation of the negatively charged dimyristoylglycerol. We also examined the amide exchange in the two bicelles. We find that despite the fact that DynA is largely unstructured in both types of bicelles, the peptide has restricted backbone dynamics, which depends on the presence of negatively charged lipids. Moreover we see that the lipid dependence is not uniform throughout the sequence, but is most noticeable for Leu5, which precedes an unusually basic stretch of amino acid residues. The findings indicate that this basic sequence may be of significance for bilayer recognition. Finally, we note that the dynamical behavior of the peptide is much more influenced by the lipid surroundings than what the structural properties are.

  • 20.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kurnik, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold2014Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, nr 3, s. 814-824Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

  • 21.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Mathias
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Analysing the morphology of DHPC/DMPC complexes by diffusion NMRManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many un-certainties remain concerning the details of the phase be-haviour of these mixtures and the morphology of the formed lipid assemblies. Here we used NMR diffusion data in com-bination with the multivariate processing method SCORE to analyze mixtures of DHPC and DMPC with the relative concentration q=[DMPC]/[DHPC]=0.5 at total lipid con-centrations from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coeffi-cients for lipid concentrations in the range 15 to 200 mM. Between 200 and 300 mM, the similar diffusion coefficients in combination with substantial signal overlap makes it difficult to get very reliable spectra and diffusion coeffi-cients with standard processing parameters, but overfactoring provided useful diffusion coefficient estimates also at these concentrations. At 50–300 mM total lipid concentration, the radii estimated from the diffusion coeffi-cient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q=0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may relia-bly be assumed to adopt the 'classical' bicelle morphology. At lower concentrations there is evidence for a more com-plex morphology with more than one type of lipid assembly in the sample.

  • 22. Bolmgren, K.
    et al.
    Vanhoenacker, Didrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Botaniska institutionen. Swedish Natural History Museum.
    Miller-Rushing, A. J.
    One man, 73 years, and 25 species. Evaluating phenological responses using a lifelong study of first flowering dates2013Ingår i: International journal of biometeorology, ISSN 0020-7128, E-ISSN 1432-1254, Vol. 57, nr 3, s. 367-375Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Phenological shifts linked to global warming reflect the ability of organisms to track changing climatic conditions. However, different organisms track global warming differently and there is an increasing interest in the link between phenological traits and plant abundance and distribution. Long-term data sets are often used to estimate phenological traits to climate change, but so far little has been done to evaluate the quality of these estimates. Here, we use a 73-year long data series of first flowering dates for 25 species from north-temperate Sweden to evaluate (i) correlations between first flowering dates and year for different time periods and (ii) linear regression models between first flowering date and mean monthly temperatures in preceding months. Furthermore, we evaluate the potential of this kind of data to estimate the phenological temperature sensitivities (i.e. number of days phenological change per degree temperature change, beta(60)) in such models. The sign of the correlations between first flowering dates and year were highly inconsistent among different time periods, highlighting that estimates of phenological change are sensitive to the specific time period used. The first flowering dates of all species were correlated with temperature, but with large differences in both the strength of the response and the period(s) of the year that were most strongly associated with phenological variation. Finally, our analyses indicated that legacy data sets need to be relatively long-term to be useful for estimating phenological temperature sensitivities (beta(60)) for inter-specific comparisons. In 10-year long observation series only one out of 24 species reached a parts per thousand yen80 % probability of estimating temperature sensitivity (beta(60)) within a +/- 1 range, and 17 out of 24 species reached a parts per thousand yen80 % probability when observation series were 20 years or shorter. The standard error for beta(60) ranged from 0.6 to 2.0 for 10-year long observation series, and 19 out of 24 species reached SE < 1 after 15 years. In general, late flowering species will require longer time series than early flowering species.

  • 23.
    Brandes, Rolf
    Stockholms universitet.
    Structure and dynamics of DNA in the solid and liquid crystalline states studied by deuterium NMR1986Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 24. Braun, Stefan
    et al.
    Mhatre, Snehit S.
    Jaussi, Marion
    Roy, Hans
    Kjeldsen, Kasper U.
    Pearce, Christof
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för geologiska vetenskaper. Aarhus University, Denmark.
    Seidenkrantz, Marit-Solveig
    Jorgensen, Bo Barker
    Lomstein, Bente Aa.
    Microbial turnover times in the deep seabed studied by amino acid racemization modelling2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 5680Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The study of active microbial populations in deep, energy-limited marine sediments has extended our knowledge of the limits of life on Earth. Typically, microbial activity in the deep biosphere is calculated by transport-reaction modelling of pore water solutes or from experimental measurements involving radiotracers. Here we modelled microbial activity from the degree of D: L-aspartic acid racemization in microbial necromass (remains of dead microbial biomass) in sediments up to ten million years old. This recently developed approach (D: L-amino acid modelling) does not require incubation experiments and is highly sensitive in stable, low-activity environments. We applied for the first time newly established constraints on several important input parameters of the D: L-amino acid model, such as a higher aspartic acid racemization rate constant and a lower cell-specific carbon content of sub-seafloor microorganisms. Our model results show that the pool of necromass amino acids is turned over by microbial activity every few thousand years, while the turnover times of vegetative cells are in the order of years to decades. Notably, microbial turnover times in million-year-old sediment from the Peru Margin are up to 100-fold shorter than previous estimates, highlighting the influence of microbial activities on element cycling over geologic time scales.

  • 25. Brown, Christian
    et al.
    Szpryngiel, Scarlett
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kuang, Guanglin
    Srivastava, Vaibhav
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    McKee, Lauren S.
    Yaoquan, Tu
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bulone, Vincent
    Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases2016Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, nr 16, s. 3072-3088Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. 

  • 26.
    Bárány-Wallje, Elsa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biophysical studies of cell-penetrating peptides and of the RNR inhibitor Sml12008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Several short peptides, so called cell-penetrating peptides, have the capability to transport large hydrophilic cargos through the cell membrane. The objective is to use these peptides as drug carriers and thereby enhance the uptake of drugs into cells.

    Three different cell-penetrating peptides are characterized in this thesis. Structure and dynamics of transportan when bound to phospholipid bicelles was determined using NMR. The hydrophobic peptide transportan and its deletion analogue Tp10 both bind to lipid head-group region of the membrane as amphipathic α-helices (papers I & II) and they were found to cause leakage in vesicles (paper IV). The membrane disturbing effect is probably part of how these peptides are translocated through the cell membrane, but also an explanation to why these peptides are found to be toxic in vivo. The high degree of toxicity limits their usefulness. We however also found that the membrane disturbing effect was significantly reduced when a large hydrophilic cargo was attached, which indicates that the properties of the whole peptide-cargo complex has to be taken into account (paper IV).

    The highly charged cell-penetrating peptide penetratin is not nearly as membrane disturbing as transportan (papers III and IV). Penetratin binds preferably to negatively charged membranes by electrostatic interactions. We used several different techniques to investigate if penetratin could be translocated through membrane model systems. All experiments consistently suggested that penetratin could not be translocated into model systems. It indicates an endocytotic uptake mechanism into cells rather than a direct membrane penetration (paper III). The ribonucleotide reductase inhibitor protein Sml1 was characterized using NMR and CD spectroscopy (paper V). Three different secondary structure elements were found, in agreement with previous NMR studies, but Sml1 does not have a well defined three-dimensional structure in solution. The N-terminus includes an α-helical region between residues 4-14 and we propose that this region interacts with the C-terminal part of the protein in the monomeric form. The N-terminus is also suggested to be a dimerization interface. Dimers are formed at concentrations above 10 µM in solution. The C-terminal region of Sml1 includes an α-helix between residues 61-80 that is crucial for binding and inhibition of RNR.

  • 27. Collins, Ruairi
    et al.
    Johansson, Ann-Louise
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Karlberg, Tobias
    Markova, Natalia
    van den Berg, Susanne
    Olesen, Kenneth
    Hammarstrom, Martin
    Flores, Alex
    Schuler, Herwig
    Schiavone, Lovisa Holmberg
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Arner, Elias S. J.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 1, s. e30581-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

  • 28.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR studies of the amyloid beta-peptide2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.

  • 29.
    Davydov, Albert
    Stockholms universitet.
    Electron paramagnetic resonance and biochemical studies of red-ox properties of the diferric/radical center in mouse and Mycobacterium tuberculosis ribonucleotide reductase R2 proteins1999Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyses the reduction of all four ribonucleotides to the corresponding deoxyribonucleotides that are used by the cells for DNA synthesis. The active enzyme from mouse, Mycobacterium tuberculosis (M. tuberculosis) and aerobically grown E. coli consists of two nonidentical subunits, proteins R1 and R2, both required for enzymatic activity. The R1 protein contains the regulatory sites and the binding site for the ribonucleotide substrates whereas the R2 subunit contains a stable tyrosyl radical and an antiferromagnetically coupled _-oxo-bridged diferric center. According to the proposed reaction mechanism the radical properties of the R2 protein are transferred to the active site of the R1 protein upon binding of the substrate molecule to the R1. This transfer is proposed to propagate via a conserved chain of hydrogen-bonded amino acids within the R1 and R2 proteins. Once generated the diferric/radical center may exist in different red-ox states: active (with Fe(III)Fe(III) center and tyrosyl radical), met (Fe(III)Fe(III) center without tyrosyl radical), mixed-valent (Fe(II)Fe(III) center) and fully reduced (Fe(II)Fe(II) center). Admission of oxygen to the fully reduced form of an R2 protein (a so-called regeneration reaction) results in a spontaneous formation of the active form. The regeneration reaction may be one of the possible ways employed by the cell for the generation of the active enzyme and therefore the study of this reaction is important for understanding the enzyme functionality.

    The aim of this thesis is to study the red-ox transitions of the diferric/radical center in mouse and Mycobacterium tuberculosis R2 proteins. Despite the significant similarities in the structure, the red-ox properties of the diferric/radical centers in mouse and M. tuberculosis R2 proteins are significantly different. The diferric/radical center in mouse R2 was found to be much more accessible for the external reductants than the centers of E. coli and M. tuberculosis R2 proteins. A higher accessibility of the diferric/radical center in mouse R2 protein can be explained by the presence of an open channel from the surface of the protein to the diferric/radical center. The tyrosyl radical in mouse R2 protein strongly interacts with the diferric center. Removing the tyrosyl radical in the active mouse R2 protein results in irreversible structural changes of the diferric cluster leading to an inactivation of the protein. Therefore the met form of mouse R2 protein can not be stabilized. Unlike mouse R2, the tyrosyl radical of M. tuberculosis R2 protein exhibits extremely weak magnetic interaction with the diferric center. The met form of M. tuberculosis R2 can be easily obtained and stabilized by the treatment of the active enzyme with hydroxyurea. The results of the chemical reduction of the diferric/radical center in the native mouse R2 protein as well as in two mutants (D266A and Y370W) in a proposed electron transfer pathway indicate that in all cases the second order rate constants in the mutants are comparable or faster than in native protein suggesting that the proposed radical transfer pathway is not important for the chemical reduction to proceed.

    Studying the oxidation of the fully reduced mouse R2 protein by different non-oxygen oxidants, we have demonstrated that the transitions between Fe(III)Fe(III), Fe(II)Fe(II) and Fe(II)Fe(III) states of this protein are fully reversible. The possibility to form a proper binuclear iron center without using molecular oxygen as an oxidant suggests that the _-oxo-bridge in mouse R2 protein does not necessary need molecular oxygen to be formed. Application of the low temperature reduction to mouse and M. tuberculosis R2 proteins demonstrated a presence of two structurally different diferric clusters giving rise to two distinct mixed-valent EPR signals. Whereas the shape of the mixed-valent EPR signal generated by _-irradiation at 77 K in mouse R2 protein is significantly affected by the presence of the tyrosyl radical, we did not observe any effect of the tyrosyl radical presence on the shape of mixed - valent signal generated in M. tuberculosis R2.

  • 30.
    Dicker, Andrea
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    On the correlation between in-vitro and in-vivo estimates of the thermogenic capacity of brown adipose tissue1995Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 31.
    Dou, Dan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    da Silva, Diogo V.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordholm, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wang, Hao
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daniels, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion2014Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, nr 21, s. 3363-3374Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins. On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N-out-C-in HA and M2 TMDs but not the N-in-C-out TMDs from the type II membrane protein neuraminidase (NA). To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes. Our results show that the marginally hydrophobic NA TMDs (Delta G(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once similar to 70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by similar to 100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins. Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.

  • 32. El-Sayed, R.
    et al.
    Waraky, A.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Albabtain, R.
    Eigammal, K.
    Shityakov, S.
    Muhammed, M.
    Hassan, M.
    Degradation of pristine and oxidized single wall carbon nanotubes by CYP3A42019Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 515, nr 3, s. 487-492Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Carbon nanotubes (CNTs) are a class of carbon based nanomaterials which have attracted substantial attention in recent years as they exhibit outstanding physical, mechanical and optical properties. In the last decade many studies have emerged of the underlying mechanisms behind CNT toxicity including malignant transformation, the formation of granulomas, inflammatory responses, oxidative stress, DNA damage and mutation. In the present investigation, we studied the biodegradation of single-walled carbon nanotubes (SWCNTs) by Cytochrome P450 enzymes (CYP3A4) through using Raman spectroscopy. CYP3A4 is known isozyme accountable for metabolizing various endogenous and exogenous xenobiotics. CYP3A4 is expressed dominantly in the liver and other organs including the lungs. Our results suggest that CYP3A4 has a higher affinity for p-SWNTs compared to c-SWNTs. HEK293 cellular viability was not compromised when incubated with SWNT. However, CYP3A4 transfected HEK293 cell line showed no digestion of cSWNTs after incubation for 96 h. Cellular uptake of c-SWNTs was observed by electron microscopy and localization of c-SWNTs was confirmed in endosomal vesicles and in the cytoplasm. This is the first study CYP3A4 degrading both p-SWNTs and c-SWNTs in an in vitro setup. Interestingly, our results show that CYP3A4 is more proficient in degrading p-SWNTs than c-SWNTs. We also employed computational modeling and docking assessments to develop a further understanding of the molecular interaction mechanism.

  • 33.
    Elías-Wolff, Federico
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A computational approach to curvature sensing in lipid bilayers2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses the local curvature gradient, and not just the point-wise local curvature. Finally, dispensing with the buckling methodology, we simulated a series of symmetric transmembrane multimers embedded in cylindrical bilayers. Based on the results of these simulations and theoretical arguments, we discuss the relationship between structural symmetry and curvature sensitivity. We conclude that anisotropic (i.e. orientation-dependent) curvature sensing is strongly limited by odd and high order rotational symmetries. However, measurements of in-plane orientation on peptides and asymmetric proteins, as well as dimers and tetramers, should yield valuable information. Our method, along with our initial conclusions, provides an useful tool for the understanding of the relationship between membrane shape and membrane protein function, and should prove useful to biophysicists in the design and interpretation of experimental curvature sensing assays.

  • 34.
    Elías-Wolff, Federico
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Lindén, Martin
    Lyubartsev, Alexander P.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Brandt, Erik G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling2018Ingår i: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 14, nr 3, s. 1643-1655Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

  • 35.
    Elías-Wolff, Federico
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Lindén, Martin
    Lyubartsev, Alexander P.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Brandt, Erik G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Curvature sensing by cardiolipin in simulated buckled membranesIngår i: Artikel i tidskrift (Refereegranskat)
  • 36.
    Emanuelsson, Olof
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Large-scale prediction of protein subcellular localization: sequence-based methods and applications2002Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 37.
    Eremina, Nadejda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Infrared spectroscopic studies: from small molecules to large2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Infrared light (IR) was first discovered by Friedrich Wilhelm Herschel in 1800. However, until 1940’s, molecular IR studies involved only water and small organic molecules, because of the long measurement times. Development Fourier transform infrared spectroscopy (FTIR) has minimized the time required to obtain data, making it possible to investigate bigger biological systems, e.g. proteins and nucleic acids.This thesis concentrates on the applications of different IR spectroscopic techniques to a variety of biological systems and development of new approaches to study complicated biological events.

    The first paper in this work concerns using so-called caged compounds to study the aggregation of Alzheimer’s Aβ-peptide which is linked to the formation of neurotoxic fibrils in the brain. By adding caged-sulfate to the Aβ samples we were able to change the pH of the sample, while recording IR data and study fibril formation in a time-resolved manner. Then we used caged–ADP to study the production of ATP and creatine, mediated by creatine kinase (CK). Using CK as a helper enzyme we studied the effects of the phosphate binding on the secondary structure of SR Ca2+ATPse and determined the structural differences between two similar states Ca2E1ADP and Ca2E1ATP.

    In the second part of the thesis we used ATR-FTIR spectroscopy and a specially designed dialysis setup, to develop a general method to detect ligand binding events by observing the IR absorbance changes in the water hydration shell around the molecules. The same method was used to determine the binding of DNA to the transcription factors of the E2F family. E2F proteins play main part in the gene regulatory networks that control cell development. However how they recognize their DNA-binding sites and the mechanism of binding is not well understood. By using ATR-FTIR, we observed the changes in the secondary structure of the proteins, as well as the distortions to the DNA upon E2F-DNA complex formation.

  • 38.
    Eremina, Nadejda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Interaction between Transcription Factors of E2F family and DNA Studied with Infrared SpectroscopyManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The E2F transcription factors are crucial for the regulation of genes required for proper progression through the cell cycle. Since E2Fs can initiate both cell proliferation and cell death, it is not surprising that these proteins are the subjects of extensive studies. In this work we characterize the formation of E2F1- and E2F8-DNA complexes with Fourier transform infrared spectroscopy. We demonstrate the changes in the secondary structure of the E2F1 and E2F8, in particular the disappearance of regular α-helices. We also show the perturbation to the DNA double helix and characterize the interactions between the amino acids in the proteins and the bases in the DNA.

  • 39.
    Ezzat, Kariem
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Karolinska Institutet, Sweden.
    Pernemalm, Maria
    Pålsson, Sandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Roberts, Thomas C.
    Järver, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Dondalska, Aleksandra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Bestas, Burcu
    Sobkowiak, Michal J.
    Levanen, Bettina
    Skold, Magnus
    Thompson, Elizabeth A.
    Saher, Osama
    Kari, Otto K.
    Lajunen, Tatu
    Ekström, Eva Sverremark
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nilsson, Caroline
    Ishchenko, Yevheniia
    Malm, Tarja
    Wood, Matthew J. A.
    Power, Ultan F.
    Masich, Sergej
    Linden, Anders
    Sandberg, Johan K.
    Lehtio, Janne
    Spetz, Anna-Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    EL Andaloussi, Samir
    The viral protein corona directs viral pathogenesis and amyloid aggregation2019Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, artikel-id 2331Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

  • 40.
    Fransén, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Biophysical simulation of cortical associative memory1996Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 41. Gadalla, Salah-Eldin
    et al.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lara Vasquez, Patricia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ericsson, Christer
    Zhao, Jian
    Nister, Monica
    EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells2013Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 439, nr 2, s. 203-208Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell marker and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

  • 42.
    González-Bengtsson, Amanda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    ELOVL2 and PUFA biosynthesis: Impact on sex-specific processes in mammals2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Endogenously synthesized PUFAs (Polyunsaturated Fatty Acids) vary in production depending on medical conditions, gender, developmental phase, age, fertility status, pregnancy and lactation. PUFA biosynthesis is further regulated via amount of dietary intake as well as genetically. A key player in the PUFA biosynthesis enzyme machinery is the fatty acid elongase Elongation of very long-chain fatty acids 2 (ELOVL2) that is essential for the production of the omega-3 PUFA docosahexaenoic acid (DHA, 22:6 n-3) that has many beneficial health effects. In the omega-6 PUFA series, ELOVL2 produces docosapentaenoic acid (DPA n-6, 22:5 n-6). Specifically, ELOVL2 enables the elongation of PUFA with 22 carbons to generate precursors of 24 carbons for the generation of the previously mentioned end products as well as very long-chain (VLC) PUFA up to C34.

    This thesis elucidates that estrogen has the power to modulate ELOVL2 expression in breast cancer cells and that this probably is due to ligand dependent binding of the estrogen receptor alpha (ERα) to the Elovl2 enhancer. In addition, estrogen via ERα modulates hepatic levels of Elovl2 in mice in a gender specific manner. Ablation of Elovl2 leads to whole body deficiency of DHA. The thesis unravels that systemic DHA levels in neonatal mice is determined both by the ELOVL2 status of the mother and the offspring. Ablation of Elovl2 also leads to impaired spermatogenesis and infertility in male mice. However, heterozygous Elovl2-ablated mice, differ in fertility potency depending on strain. Here we show that this discrepancy is linked to an altered ratio between saturated and mono-unsaturated fatty acids and VLC-PUFA moieties of glucosylceramides in testis.

  • 43.
    Griese, Julia J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Uppsala University, Sweden.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD2019Ingår i: acta crystallographica section d structural biology, ISSN 2059-7983, Vol. 75, s. 764-771Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

  • 44.
    Griese, Julia J.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    X ray reduction correlates with soaking accessibility as judged from four non crystallographically related diiron sites2012Ingår i: METALLOMICS, ISSN 1756-5901, Vol. 4, nr 9, s. 894-898Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.

  • 45.
    Gustafsson, Anders
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Development of a versatile algorithm for optimization of radiation therapy1996Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 46.
    Gómez-Llobregat, Jordi
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elías-Wolff, Federico
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindén, Martin
    Anisotropic Membrane Curvature Sensing by Amphipathic Peptides2016Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, nr 1, s. 197-204Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many proteins and peptides have an intrinsic capacity to sense and induce membrane curvature, and play crucial roles for organizing and remodeling cell membranes. However, the molecular driving forces behind these processes are not well understood. Here, we describe an approach to study curvature sensing by simulating the interactions of single molecules with a buckled lipid bilayer. We analyze three amphipathic antimicrobial peptides, a class of membrane-associated molecules that specifically target and destabilize bacterial membranes, and find qualitatively different sensing characteristics that would be difficult to resolve with other methods. Our findings provide evidence for direction-dependent curvature sensing mechanisms in amphipathic peptides and challenge existing theories of hydrophobic insertion. The buckling approach is generally applicable to a wide range of curvature-sensing molecules, and our results provide strong motivation to develop new experimental methods to track position and orientation of membrane proteins.

  • 47.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts.

    The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

  • 48. Heffeter, P.
    et al.
    Popovic-Bijelic, Ana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Saiko, P.
    Dornetshuber, R.
    Jungwirth, U.
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biglino, D.
    Jakupec, M. A.
    Elbling, L.
    Micksche, M.
    Szekeres, T.
    Keppler, B. K.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berger, W.
    Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)2009Ingår i: Current Cancer Drug Targets, ISSN 1568-0096, E-ISSN 1873-5576, Vol. 9, nr 5, s. 595-607Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

  • 49.
    Hertz, John A.
    et al.
    Stockholms universitet, Nordiska institutet för teoretisk fysik (Nordita).
    Roudi, Yasser
    Stockholms universitet, Nordiska institutet för teoretisk fysik (Nordita).
    Tyrcha, Joanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen.
    Ising model for inferring network structure from spike data2013Ingår i: Principle of Neural Coding / [ed] Rodrigo Quian Quiroga, Stefano Panzeri, Boca/Raton: CRC Press, 2013, s. 527-546Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Now that spike trains from many neurons can be recorded simultaneously, there is a need for methods to decode these data to learn about the networks that these neurons are part of. One approach to this problem is to adjust the parameters of a simple model network to make its spike trains resemble the data as much as possible. The connections in the model network can then give us an idea of how the real neurons that generated the data are connected and how they influence each other. In this chapter we describe how to do this for the simplest kind of model: an Ising network. We derive algorithms for finding the best model connection strengths for fitting a given data set, as well as faster approximate algorithms based on mean field theory. We test the performance of these algorithms on data from model networks and experiments.

  • 50.
    Hertz, John
    et al.
    Nordita.
    Roudi, Yasser
    Nordita.
    Tyrcha, Joanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen.
    Ising models for inferring network structure from spike data2011Ingår i: Principle of Neural Coding / [ed] S. Panzeri, R.Q. Quiroga, Cornell University Press, 2011Kapitel i bok, del av antologi (Refereegranskat)
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