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  • 1.
    Abelein, Axel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bolognesi, Benedetta
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lendel, Christofer
    Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 1, s. 126-137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

  • 2. Adase, Christopher A.
    et al.
    Draheim, Roger R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Manson, Michael D.
    The Residue Composition of the Aromatic Anchor of the Second Transmembrane Helix Determines the Signaling Properties of the Aspartate/Maltose Chemoreceptor Tar of Escherichia coli2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 9, s. 1925-1932Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was: present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.

  • 3. Adase, Christopher A.
    et al.
    Draheim, Roger R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Goethe University .
    Rueda, Garrett
    Desai, Raj
    Manson, Michael D.
    Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the Tar(Ec) Chemoreceptor2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 16, s. 2729-2738Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia colt aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild type Tar(Ec) is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six residue sequence at positions 207-212 Moving various combinations of aromatic residues among these positions generates substantial changes M receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.

  • 4.
    Adrait, Annie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öhrström, Maria
    Barra, Anne-Laure
    The High Field Laboratory, CNRS/MPI, Grenoble, France.
    Thelander, Lars
    Department of Medical Biochemistry and Biophysicis, Umeå University.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse2002Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 20, s. 6510-6516Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the μ-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

  • 5.
    Almqvist, Jonas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för fysikalisk kemi, oorganisk kemi och strukturkemi.
    Huang, Yafei
    Hovmöller, Sven
    Wang, Da-Neng
    Homology Modeling of the Human Microsomal Glucose 6-Phosphate Transporter Explains the Mutations That Cause the Glycogen Storage Disease Type Ib2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, s. 9289-9297Artikkel i tidsskrift (Fagfellevurdert)
  • 6.
    Ariöz, Candan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    al Bakali, Amin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liebau, Jobst
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Götzke, Hansjörg
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 33, s. 5533-5544Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

  • 7.
    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 Variants2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 24, s. 4157-4167Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 8.
    Blasco, Pilar
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Patel, Dhilon S.
    Engström, Olof
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Im, Wonpil
    Widmalm, Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Conformational Dynamics of the Lipopolysaccharide from Escherichia coli O91 Revealed by Nuclear Magnetic Resonance Spectroscopy and Molecular Simulations2017Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 29, s. 3826-3839Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The outer leaflet of the outer membrane in Gram-negative bacteria contains lipopolysaccharides (LPS) as a major component, and the outer membrane provides a physical barrier and protection against hostile environments. The enterohemorrhagic Escherichia coli of serogroup O91 has an O-antigen polysaccharide (PS) with five sugar residues in the repeating unit (RU), and the herein studied O-antigen PS contains similar to 10 RUs. H-1-C-13 HSQC-NOESY experiments on a 1-C-13-labeled PS were employed to deduce H-1-H-1 cross-relaxation rates and transglycosidic (3)J(CH) related to the psi torsional angles were obtained by H-1-H-1 NOESY experiments. Dynamical parameters were calculated from the molecular dynamics (MD) simulations of the PS in solution and compared to those from C-13 nuclear magnetic resonance (NMR) relaxation studies. Importantly, the MD simulations can reproduce the dynamical behavior of internal correlation times along the PS chain. Two-dimensional free energy surfaces of glycosidic torsion angles delineate the conformational space available to the O-antigen. Although similar with respect to populated states in solution, the O-antigen in LPS bilayers has more extended chains as a result of spatial limitations due to close packing. Calcium ions are highly abundant in the phosphate-containing core region mediating LPS LPS association that is crucial for maintaining bilayer integrity, and the negatively charged O-antigen promotes a high concentration of counterbalancing potassium ions. The ensemble of structures present for the PS in solution is captured by the NMR experiments, and the similarities between the O-antigen on its own and as a constituent of the full LPS in a bilayer environment make it possible to realistically describe the LPS conformation and dynamics from the MD simulations.

  • 9.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Active Site Midpoint Potentials in Different Cytochrome c Oxidase Families: A Computational Comparison2019Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, nr 15, s. 2028-2038Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain, reducing molecular oxygen to water. The binuclear active site in CcO comprises a high-spin heme associated with a Cu-B complex and a redox active tyrosine. The electron transport in the respiratory chain is driven by increasing midpoint potentials of the involved cofactors, resulting in a release of free energy, which is stored by coupling the electron transfer to proton translocation across a membrane, building up an electrochemical gradient. In this context, the midpoint potentials of the active site cofactors in the CcOs are of special interest, since they determine the driving forces for the individual oxygen reduction steps and thereby affect the efficiency of the proton pumping. It has been difficult to obtain useful information on some of these midpoint potentials from experiments. However, since each of the reduction steps in the catalytic cycle of oxygen reduction to water corresponds to the formation of an O-H bond, they can be calculated with a reasonably high accuracy using quantum chemical methods. From the calculated O-H bond strengths, the proton-coupled midpoint potentials of the active site cofactors can be estimated. Using models representing the different families of CcO's (A, B, and C), the calculations give midpoint potentials that should be relevant during catalytic turnover. The calculations also suggest possible explanations for why some experimentally measured potentials deviate significantly from the calculated ones, i.e., for Cu-B in all oxidase families, and for heme b(3) in the C family.

  • 10.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Can Reduction of NO to N2O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans-Mechanism?2017Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 1, s. 120-131Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    As part of microbial denitrification, NO is reduced to N2O in the membrane bound enzyme nitric oxide reductase, NOR The N N coupling occurs in the diiron binuclear active site, BNC, and different mechanisms for this reaction step have been suggested. Computational studies have supported a so-called cis:b(3)-mechanism, in which the hyponitrite product of the reductive N N bond formation coordinates with one nitrogen to the heme iron and with both oxygens to the non-heme iron in the BNC. In contrast, experimental results have been interpreted to support a so-called trans-mechanism, in which the hyponitrite intermediate coordinates with one nitrogen atom to each of the two iron ions. Hybrid density functional theory is used here to perform an extensive search for possible intermediates of the NO reduction in the cNOR enzyme. It is found that hyponitrite structures coordinating with their negatively charged oxygens to the positively charged iron ions are the most stable ones. The hyponitrite intermediate involved in the suggested trans-mechanism, which only coordinates with the nitrogens to the iron ions, is found to be prohibitively high in energy, leading to a too slow reaction, which should rule out this mechanism. Furthermore, intermediates binding one NO molecule to each iron ion in the BNC, which have been suggested to initiate the trans-mechanism, are found to be too high in energy to be observable, indicating that the experimentally observed electron paramagnetic resonance signals, taken to support such an iron-nitrosyl dimer intermediate, should be reinterpreted.

  • 11.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 3, s. 489-500Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytochrome c oxidase, the terminal enzyme in the respiratory chain, reduces molecular oxygen to water and stores the released energy through electrogenic chemistry and proton pumping across the membrane. Apart from the heme-copper binuclear center, there is a conserved tyrosine residue in the active site (BNC). The tyrosine delivers both an electron and a proton during the O-O bond cleavage step, forming a tyrosyl radical. The catalytic cycle then occurs in four reduction steps, each taking up one proton for the chemistry (water formation) and one proton to be pumped. It is here suggested that in three of the reduction steps the chemical proton enters the center of the BNC, leaving the tyrosine unprotonated with radical character. The reproprotonation of the tyrosine occurs first in the final reduction step before binding the next oxygen molecule. It is also suggested that this reduction mechanism and the presence of the tyrosine are essential for the proton pumping. Density functional theory calculations on large cluster models of the active site show that only the intermediates with the proton in the center of the BNC and with an unprotonated tyrosyl radical have a high electron affinity of similar size as the electron donor, which is essential for the ability to take up two protons per electron and thus for the proton pumping. This type of reduction mechanism is also the only one that gives a free energy profile in accordance with experimental observations for the amount of proton pumping in the working enzyme.

  • 12.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Siegbahn, Per E. M.
    Mechanism for N2O Generation in Bacterial Nitric Oxide Reductase: A Quantum Chemical Study2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 25, s. 5173-5186Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The catalytic mechanism of reduction of NO to N2O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N2O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.

  • 13. Busenlehner, Laura
    et al.
    Brändén, Gisela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Namslauer, Ida
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Armstrong, Richard
    Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 1, s. 73-83Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples' the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa<sub>3</sub>), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O<sub>2</sub> reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains ∼1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton transtocation by CytcO.

  • 14.
    Dourado, Daniel F. A. R.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 45, s. 8069-8078Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 15. Edvardsson, Anna
    et al.
    Shapiguzov, Alexey
    Petersson, Ulrika A.
    Stockholms universitet.
    Schroder, Wolfgang P.
    Vener, Alexander V.
    Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-22007Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 33, s. 9432-9442Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.

  • 16.
    Esbjörner, Elin K.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oglęcka, Kamila
    Lincoln, Per
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordén, Bengt
    Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models2007Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 47, s. 13490-13504Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue

  • 17. Jemth, Ann-Sofie
    et al.
    Scaletti, Emma
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Carter, Megan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Helleday, Thomas
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Lund University, Sweden.
    Crystal Structure and Substrate Specificity of the 8-oxo-dGTP Hydrolase NUDT1 from Arabidopsis thaliana2019Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, nr 7, s. 887-899Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Arabidopsis thaliana NUDT1 (AtNUDT1) belongs to the Nudix family of proteins, which have a diverse range of substrates, including oxidized nucleotides such as 8-oxo-dGTP. The hydrolysis of oxidized dNTPs is highly important as it prevents their incorporation into DNA, thus preventing mutations and DNA damage. AtNUDT1 is the sole Nudix enzyme from A. thaliana shown to have activity against 8-oxo-dGTP. We present the structure of AtNUDT1 in complex with 8-oxo-dGTP. Structural comparison with bacterial and human homologues reveals a conserved overall fold. Analysis of the 8-oxo-dGTP binding mode shows that the residues Asn76 and Ser89 interact with the 08 atom of the substrate, a feature not observed in structures of protein homologues solved to date. Kinetic analysis of wild-type and mutant AtNUDT1 confirmed that these active site residues influence 8-oxo-dGTP hydrolysis. A recent study showed that AtNUDT1 is also able to hydrolyze terpene compounds. The diversity of reactions catalyzed by AtNUDT1 suggests that this Nudix enzyme from higher plants has evolved in a manner distinct to those from other organisms.

  • 18.
    Johansson, Ann-Louise
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Carlsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hosler, Jonathan P
    Gennis, Robert B
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton Uptake and pK(a) Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 5, s. 827-836Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O-2 to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located similar to 10 angstrom from the protein surface, is particularly sensitive to mutations that uncouple the O-2 reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

  • 19. Keller, Rebecca
    et al.
    Ariöez, Candan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hansmeier, Nicole
    Stenberg-Bruzell, Filippa
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Burstedt, Malin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vikström, David
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kelly, Amelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hunke, Sabine
    The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties2015Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 23, s. 3670-3676Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.

  • 20.
    Lagerstedt, Jens
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reeve, Ian
    Voss, John C
    Persson, Bengt L
    The structure and function of GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae2005Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 2, s. 511-517Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Δgtr1 similar to the Δpho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84−green fluorescent protein or Pho84−myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1−185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186−310) displayed no evidence of conformational changes upon GTP addition.

  • 21. Lendel, Christofer
    et al.
    Bolognesi, Benedetta
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Detergent-like interaction of Congo red with the amyloid beta peptide2010Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, nr 7, s. 1358-1360Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimer's disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.

  • 22.
    Lepp, Håkan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Svahn, Emelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Faxén, Kristina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 17, s. 4929-4935Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytochrome c oxidase couples electron transfer from cytochrome C to 02 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with 02, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.

  • 23.
    Liao, Rong-Zhen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Georgieva, Polina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Yu, Jian-Guo
    Himo, Fahmi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Mechanism of mycolic acid cyclopropane synthase: A theoretical study2011Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, nr 9, s. 1505-1513Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.

  • 24.
    Lill, Sten O. Nilsson
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    An Autocatalytic Mechanism for NiFe-Hydrogenase: Reduction to Ni(I) Followed by Oxidative Addition2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 5, s. 1056-1066Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mechanism for H-2 cleavage in NiFe-hydrogenase has been reinvestigated with large models using both hybrid DFT by itself, or in a QM/MM scheme following the ONIOM approach. Heterolytic cleavage, with one hydrogen ending up as a bridging hydride and one as a proton on a cysteine ligand, was found to have a barrier slightly too high to be compatible with measured catalytic turnover rates. Alternative mechanisms were therefore investigated. In the finally suggested mechanism, heterolytic cleavage is used only as an initial step to generate a complex with nickel in oxidation state Ni(I). In the following cycles, H-2 is instead cleaved on nickel using an oxidative addition mechanism with a lower barrier. It was found that the ONIOM results for the reaction mechanism in NiFe-hydrogenase needed to be corrected by large model DFT results to be more reliable. This was mainly an effect of overestimation of polarization effects of the QM region by the MM region due to the particular treatment of the electrostatic interactions and the use of a standard (nonpolarizable) force field.

  • 25.
    Lind, Jesper
    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 Interactions of Dynorphins2006Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, s. 15931-15940Artikkel i tidsskrift (Fagfellevurdert)
  • 26.
    Lind, Jesper
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rämö, Tuulia
    Rosén Klement, Maria L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bárány-Wallje, Elsa
    Epand, Richard M.
    Epand, Raquel F.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase2007Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 19, s. 5664-5677Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

  • 27.
    Lindström, Ida
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dogan, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement2017Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 32, s. 4145-4153Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Phi-value analysis) and found that the native protein protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the (Phi-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation condensation mechanism. Thus, native hydrophobic binding interactions at the rate limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

  • 28. Luo, Jinghui
    et al.
    Wärmlander, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abrahams, Jan Pieter
    Alzheimer Peptides Aggregate into Transient Nanoglobules That Nucleate Fibrils2014Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 40, s. 6302-6308Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Protein/peptide oligomerization, cross-beta strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer's amyloid beta (A beta) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the A beta peptides coalesced into apparently unstructured globules (15-200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately >= 100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-beta strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation.

  • 29. Martyna, Agnieszka
    et al.
    Gomez-Llobregat, Jordi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindén, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rossman, Jeremy S.
    Curvature Sensing by a Viral Scission Protein2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 25, s. 3493-3496Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Membrane scission is the final step in all budding processes wherein a membrane neck is sufficiently constricted so as to allow for fission and the release of the budded particle. For influenza viruses, membrane scission is mediated by an amphipathic helix (AH) domain in the viral M2 protein. While it is known that the M2AH alters membrane curvature, it is not known how the protein is localized to the center neck of budding virions where it would be able to cause membrane scission. Here, we use molecular dynamics simulations on buckled lipid bilayers to show that the M2AH senses membrane curvature and preferentially localizes to regions of high membrane curvature, comparable to that seen at the center neck of budding influenza viruses. These results were then validated using in vitro binding assays to show that the M2AH senses membrane curvature by detecting lipid packing defects in the membrane. Our results show that the M2AH senses membrane curvature and suggest that the AH domain may localize the protein at the viral neck where it can then mediate membrane scission and the release of budding viruses.

  • 30.
    Narwal, Mohit
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jemth, Ann-Sofie
    Gustafsson, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Almlöf, Ingrid
    Warpman Berglund, Ulrika
    Helleday, Thomas
    Stenmark, Pål
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity2018Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, nr 5, s. 593-603Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to non-malignant cells making cancer cells more dependent on MTH1 for survival. The possibility to specifically target cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. Progression of MTH1 inhibitors into the clinic requires animal studies and knowledge about species differences in potency of inhibitors are of vital importance. We here show that the human MTH1 inhibitor TH588 is approximately twenty fold less potent for inhibition of mouse MTH1 compared to human, rat, pig, and dog MTH1. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant for TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.

  • 31.
    Näsvik Öjemyr, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Faxén, Kristina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Svahn, Emelie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The membrane modulates internal proton transfer in cytochrome c oxidase2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, s. 1092-1100Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O2 reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pKa in the pH dependence profile of the proton-uptake rate. This pKa change resulted in decreased proton-uptake rates in the pH range of 6.5–9.5, which is explained in terms of lowering of the pKa of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal CuA–heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.

  • 32.
    Palombo, Isolde
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rapp, Mikaela
    Why Is the GMN Motif Conserved in the CorA/Mrs2/Alr1 Superfamily of Magnesium Transport Proteins?2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 28, s. 4842-4847Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Members of the CorA/Mrs2/Alr1 superfamily of transport proteins mediate magnesium uptake in all kingdoms of life. Family members have a low degree of sequence conservation but are characterized by a conserved extracellular loop. While the degree of sequence conservation in the loop deviates to some extent between family members, the GMN family signature motif is always present. Structural and functional data imply that the loop plays a central role in magnesium selectivity, and recent biochemical data suggest it is crucial for stabilizing the pentamer in the magnesium-free (putative open) conformation. In this study, we present a detailed structure-function analysis of the extracellular loop of CorA from Thermotoga maritima, which provides molecular insight into how the loop mediates these two functions. The data show that loop residues outside of the GMN motif can be substituted if they support the pentameric state, but the residues of the GMN motif are intolerant to substitution. We conclude that G(312) is absolutely required for magnesium uptake, M-313 is absolutely required for pentamer integrity in the putative open conformation, and N-314 plays a role in both of these functions. These observations suggest a molecular reason why the GMN motif is conserved throughout the CorA/Mrs2/Alr1 superfamily.

  • 33.
    Popović-Bijelić, Ana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Voevodskaya, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Domkin, Vladimir
    Thelander, Lars
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 27, s. 6532-6539Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

  • 34. Pratt, James R
    et al.
    Mouillon, Jean-Marie
    Lagerstedt, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pattison-Granberg, Johanna
    Lundh, Kent I
    Persson, Bengt L
    The effects of methylphosphonate, a phosphate analog, on the expression and degradation of the highaffinity phosphate transporter Pho84, in Saccharomyces cerevisiae2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 45, s. 14444-14453Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Δpho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by 31P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.

  • 35.
    Ranganathan, Anirudh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stanford University, USA.
    Dror, Ron O.
    Carlsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stanford University, USA .
    Insights into the Role of Asp79(2.50) in beta(2) Adrenergic Receptor Activation from Molecular Dynamics Simulations2014Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 46, s. 7283-7296Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Achieving a molecular-level understanding of G-protein-coupled receptor (GPCR) activation has been a long-standing goal in biology and could be important for the development of novel drugs. Recent breakthroughs in structural biology have led to the determination of high-resolution crystal structures for the beta(2) adrenergic receptor (beta(2)AR) in inactive and active states, which provided an unprecedented opportunity to understand receptor signaling at the atomic level. We used molecular dynamics (MD) simulations to explore the potential roles of ionizable residues in beta(2)AR activation. One such residue is the strongly conserved Asp79(2.50), which is buried in a transmembrane cavity and becomes dehydrated upon beta(2)AR activation. MD free energy calculations based on beta(2)AR crystal structures suggested an increase in the population of the protonated state of Asp79(2.50) upon activation, which may contribute to the experimentally observed pH-dependent activation of this receptor. Analysis of MD simulations (in total >100 mu s) with two different protonation states further supported the conclusion that the protonated Asp79(2.50) shifts the conformation of the beta(2)AR toward more active-like states. On the basis of our calculations and analysis of other GPCR crystal structures, we suggest that the protonation state of Asp(2.50) may act as a functionally important microswitch in the activation of the beta(2)AR and other class A receptors.

  • 36. Rinaldo-Matthis, Agnes
    et al.
    Ahmad, Shabbir
    Wetterholm, Anders
    Lachmann, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Morgenstern, Ralf
    Häggström, Jesper Z.
    Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C-4 Synthase2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 4, s. 848-856Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human leukotriene C-4 synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C-4 (LTC4). This molecule, together with its metabolites LTD4 and LTE4, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA(4), presumably according to an S(N)2 reaction to form LTC4. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s(-1)). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K-d(GS) = 14.3 mu M). The release of the thiolate was assessed using a GSH release experiment (1.3 s(-1)). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC4 production (k(cat) = 26 s(-1)), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.

  • 37.
    Roos, Katarina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    Density Functional Theory Study of the Manganese-Containing Ribonucleotide Reductase from Chlamydia trachomatis: Why Manganese Is Needed in the Active Complex2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 9, s. 1878-1887Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The active center of Chlamydia trachomatis (Ct) ribonucleotide reductase (RNR) has been studied using B3LYP hybrid density functional theory. Class Ic Ct RNR lacks the radical-bearing tyrosine that is crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)Tyr(rad) active state in conventional class I, Ct RNR has Mn(IV)Fe(III) at the metal center of subunit H. Based on calculated (H+, e(-))-binding energies for Ct R2, iron-substituted Ct R2, and R2 from Escherichia coli (Ec), an explanation is proposed for why the enzyme needs this novel metal center. Mn(IV) is shown to be an equally strong oxidant as the tyrosyl radical in Ec R2. Fe(IV), however, is a much too strong oxidant and would therefore not be possible in the active cofactor. The structure of the catalytic center of the active state, such as protonation state and position of Mn, is discussed. Ct R2 has a different ligand structure than conventional class I R2 with a fourth Glu (like MMO) instead of three Glu and one Asp. Calculations indicate that, in the presence of Tyr, Glu at this position is less flexible than Asp, whereas with Phe both Glu and Asp are equally flexible. This may be a reason why conventional class I RNR has an Asp, while Ct R2, lacking the tyrosine, has a Glu.

  • 38.
    Sevastik, Robin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Whitman, Christian P.
    Himo, Fahmi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Reaction mechanism of cis-3-chloroacrylic acid dehalogenase: a theoretical study2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 40, s. 9641-9649Artikkel i tidsskrift (Fagfellevurdert)
  • 39.
    Sheng, Xiang
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Patskovsky, Yury
    Vladimirova, Anna
    Bonanno, Jeffrey B.
    Almo, Steven C.
    Himo, Fahmi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Raushel, Frank M.
    Mechanism and Structure of gamma-Resorcylate Decarboxylase2018Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, nr 22, s. 3167-3175Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    gamma-Resorcylate decarboxylase (gamma-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of gamma-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by gamma-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of gamma-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn2+ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of gamma-RSD from Rhizobtum sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, gamma-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to Cl of y-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of gamma-RSD from Rhizobtum sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

  • 40.
    Sjöholm, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mamedoy, Fikret
    Styring, Stenbjörn
    Spectroscopic Evidence for a Redox-Controlled Proton Gate at Tyrosine D in Photosystem II2014Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 36, s. 5721-5723Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Tyrosine D (TyrD) is one of two well-studied redox active tyrosines in Photosystem II. TyrD shows redox kinetics much slower than that of its homologue, TyrZ, and is normally present as a stable deprotonated radical (TyrD<bold>). We have used time-resolved continuous wave electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy to show that deuterium exchangeable protons can access TyrD on a time scale that is much faster (50</bold>100 times) than that previously observed. The time of H/D exchange is strongly dependent on the redox state of TyrD. This finding can be related to a change in position of a water molecule close to TyrD.

  • 41.
    Skoog, Karl
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Escherichia coli Cell Division Protein ZipA Forms Homodimers Prior to Association with FtsZ2012Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 7, s. 1407-1415Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    ZipA is an essential component of the cell division machinery in E. coil and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.

  • 42.
    Smirnova, Irina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Chang, Hsin-Yang
    von Ballmoos, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Adelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Single Mutations That Redirect Internal Proton Transfer in the ba(3) Oxidase from Thermus thermophilus2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 40, s. 7022-7030Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa(3)-type oxidases proton uptake to the catalytic site and pump site occurs simultaneously. However, with ba(3) oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa(3) oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba(3) CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba(3) CytcO uses only a single proton pathway for transfer of the substrate and pumped protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.

  • 43.
    Smirnova, Irina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Chang, Hsin-Yang
    Gennis, Robert B.
    Fee, James A.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Functional Role of Thr-312 and Thr-315 in the Proton-Transfer Pathway in ba3 Cytochrome c Oxidase from Thermus thermophilus2010Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, nr 33, s. 7033-7039Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytochrome ba3 from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba3 oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169−16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba3 and O2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 → Ser variant in the ba3 oxidase both reactions associated with proton uptake from solution, the PR → F and F → O transitions, were slowed compared to those of wild-type ba3. The observed time constants were slowed 3-fold (for PR → F, from 60 to 170 μs in the wild type) and 30-fold (for F → O, from 1.1 to 40 ms). In the Thr-315 → Val variant, the F → O transition was 5-fold slower (5 ms) than for the wild-type oxidase, whereas the PR → F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical PR → F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O2 reduction in the ba3 oxidase.

  • 44.
    Stenmark, Pål
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gurmu, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Pär
    Crystal Structure of CaiB, a Type-III CoA Transferase in Carnitine Metabolism2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 44, s. 13996-14003Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Carnitine is an important molecule in human metabolism, mainly because of its role in the transport of long-chain fatty acids across the inner mitochondrial membrane. Escherichia coli uses carnitine as a terminal electron acceptor during anaerobic metabolism. Bacteria present in our large intestine break down carnitine that is not absorbed in the small intestine. One part of this catabolic pathway is reversible and can be utilized for bioproduction of large amounts of stereochemically pure l-carnitine, which is used medically for the treatment of a variety of human diseases. Here, we present the crystal structure of the E. coli protein CaiB, which is a member of the recently identified type-III coenzyme A (CoA) transferase family and catalyzes the transfer of the CoA moiety between γ-butyrobetaine−CoA and carnitine forming carnityl-CoA and γ-butyrobetaine. This is the first protein from the carnitine metabolic pathway to be structurally characterized. The structure of CaiB reveals a spectacular fold where two monomers are interlaced to form an interlocked dimer. A molecule of the crystallization buffer bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (bis-tris) is bound in a large pocket located primarily in the small domain, and we propose that this pocket constitutes the binding site for both substrate moieties participating in the CaiB transfer reaction. The binding of CoA to CaiB induces a domain movement that closes the active site of the protein. This is the first observation of a domain movement in the type-III CoA transferase family and can play an important role in coupling substrate binding to initiation of the catalytic reaction.

  • 45. Stone, Tracy A.
    et al.
    Schiller, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Deber, Charles M.
    Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences2015Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 7, s. 1465-1473Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.

  • 46. Stone, Tracy A.
    et al.
    Schiller, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Workewych, Natalie
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Deber, Charles M.
    Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 40, s. 5772-5779Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (similar to 30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous block in the middle of the segment or scrambled throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a threshold hydrophilicity is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomaloug translocation/insertion.

  • 47.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lakovleva, Irina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Georgiev, Alexander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana2011Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, nr 21, s. 4451-4466Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

  • 48.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into the Membrane Interacting Properties of the C-Terminal Domain of the Monotopic Glycosyltransferase DGD2 in Arabidopsis thaliana2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 49, s. 6776-6786Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glycosyltransferases (GTs) are responsible for regulating the membrane composition of plants. The synthesis of one of the main lipids in the membrane, the galactolipid digalactosyldiacylglycerol, is regulated by the enzyme digalactosyldiacylglycerol synthase 2 (atDGD2) under starving conditions, such as phosphate shortage. The enzyme belongs to the GT-B fold, characterized by two beta/alpha/beta Rossmann domains that are connected by a flexible linker. atDGD2 has previously been shown to attach to lipid membranes by the N-terminal domain via interactions with negatively charged lipids. The role of the C-terminal domain in the membrane interaction is, however, not known. Here we have used a combination of in silico prediction methods and biophysical experimental techniques to shed light on the membrane interacting properties of the C-terminal domain. Our results demonstrate that there is an amphipathic sequence, corresponding to residues V240-E258, that interacts with lipids in a charge-dependent way. A second sequence was identified as being potentially important, with a high charge density, but no amphipathic character. The features of the plant atDGD2 observed here are similar in prokaryotic glycosyltransferases. On the basis of our results, and by analogy to other glycosyltransferases, we propose that atDGD2 interacts with the membrane through the N-terminus and with parts of the C-terminus acting as a switch, allowing for a dynamic interaction with the membrane.

  • 49.
    Tiiman, Ann
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. The National Institute of Chemical Physics and Biophysics, Estonia.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vukojevic, Vladana
    Heterogeneity and Turnover of Intermediates during Amyloid-beta (A beta) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy2015Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, nr 49, s. 7203-7211Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Self-assembly of amyloid beta (A beta) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among A beta molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. A beta self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding A beta aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying A beta aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of A beta peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 x 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during A beta aggregation. The effect of different A beta concentrations, A beta:ThT ratios, differences between the 40 (A beta 40) and 42 (A beta 42) residue long variants of A beta, and the effect of stirring were also examined.

  • 50.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Papadopoulos, Evangelos
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 25, s. 5813-5821Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

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