| 1. |
- Awad, Wael, et al.
(författare)
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Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 57:7, s. 1166-1177
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.
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| 2. |
- Blasco, Pilar, et al.
(författare)
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Conformational Dynamics of the Lipopolysaccharide from Escherichia coli O91 Revealed by Nuclear Magnetic Resonance Spectroscopy and Molecular Simulations
- 2017
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 56:29, s. 3826-3839
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Blikstad, Cecilia, et al.
(författare)
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Learning to Build a β-Carboxysome
- 2019
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 58:16, s. 2091-2092
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Forskningsöversikt (refereegranskat)
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| 4. |
- Blomberg, Margareta R. A.
(författare)
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Active Site Midpoint Potentials in Different Cytochrome c Oxidase Families : A Computational Comparison
- 2019
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 58:15, s. 2028-2038
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Blomberg, Margareta R. A.
(författare)
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Can Reduction of NO to N2O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans-Mechanism?
- 2017
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 56:1, s. 120-131
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Blomberg, Margareta R. A.
(författare)
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Mechanism of Oxygen Reduction in Cytochrome c Oxidase and the Role of the Active Site Tyrosine
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:3, s. 489-500
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Clark, Andrew G., et al.
(författare)
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Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation
- 2018
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 57:5, s. 614-619
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Tidskriftsartikel (refereegranskat)abstract
- Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (δ,δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (δ,δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for δ,δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, δ,δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that δ,δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.
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| 8. |
- Danelius, Emma, et al.
(författare)
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Halogen Bonding: A Powerful Tool for Modulation of Peptide Conformation
- 2017
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 56:25, s. 3265-3272
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Tidskriftsartikel (refereegranskat)abstract
- Halogen bonding is a weak chemical force that has so far mostly found applications in crystal engineering. Despite its potential for use in drug discovery, as a new molecular tool in the direction of molecular recognition events, it has rarely been assessed in biopolymers. Motivated by this fact, we have developed a peptide model system that permits the quantitative evaluation of weak forces in a biologically relevant proteinlike environment and have applied it for the assessment of a halogen bond formed between two amino acid side chains. The influence of a single weak force is measured by detection of the extent to which it modulates the conformation of a cooperatively folding system. We have optimized the amino acid sequence of the model peptide on analogues with a hydrogen bond-forming site as a model for the intramolecular halogen bond to be studied, demonstrating the ability of the technique to provide information about any type of weak secondary interaction. A combined solution nuclear magnetic resonance spectroscopic and computational investigation demonstrates that an interstrand halogen bond is capable of conformational stabilization of a beta-hairpin foldamer comparable to an analogous hydrogen bond. This is the first report of incorporation of a conformation-stabilizing halogen bond into a peptide/protein system, and the first quantification of a chlorine-centered halogen bond in a biologically relevant system in solution.
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| 9. |
- Dimitroff, George, et al.
(författare)
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(1,3;1,4)-beta-Glucan Biosynthesis by the CSLF6 Enzyme : Position and Flexibility of Catalytic Residues Influence Product Fine Structure
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:13, s. 2054-2061
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-beta-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4) -linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-beta-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-beta-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.
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| 10. |
- Dogan, Jakob, et al.
(författare)
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Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:37, s. 5289-5295
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Tidskriftsartikel (refereegranskat)abstract
- Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data. demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (similar to 10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured State. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein protein interactions.
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