| 1. |
- Allgardsson, Anders, et al.
(författare)
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Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:20, s. 5514-5519
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Tidskriftsartikel (refereegranskat)abstract
- Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.
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| 2. |
- Alriksson, Björn, et al.
(författare)
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Fish feed from wood
- 2014
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Ingår i: Cellulose Chemistry and Technology. - 0576-9787. ; 48, s. 843-848
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Tidskriftsartikel (refereegranskat)abstract
- Increased demand of fish in combination with overexploitation of the fish stocks of the oceans has led to an increased production of fish through aquaculture. Today, fishmeal is the main protein source in fish feed for most aquaculture species. However, fishmeal is soon expected to fall short of demand as its production is associated with environmental problems. This shortage must therefore be met by sustainable alternative protein sources. Protein-rich microorganisms (i.e. Single cell protein) is an interesting option as a fishmeal substitute in fish feed which, in addition, can be produced as an important co-product in wood-based biorefineries. In the current study, four different microorganisms were cultivated on five different residual streams from Swedish wood-based biorefineries. Screening experiments were carried out in shake flasks, optimization experiments in benchtop bioreactors, and scale-up experiments were performed in a 50-litre pilot bioreactor. In addition, a demo-scale experiment was carried out in the Swedish Biorefinery Demo Plant. Microbial biomass from the scale-up experiments was collected and used for production of different fish feed formulations which, in turn, were used in feeding trials of the freshwater fish Tilapia. Fishes fed with feed, in which part of the fishmeal had been substituted with Single cell protein, showed similar or better growth than fishes fed with a fishmeal-based control feed.
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| 3. |
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| 4. |
- Donev, Evgeniy N., et al.
(författare)
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Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners
- 2023
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7644 .- 1467-7652. ; 21:5, s. 1005-1021
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Tidskriftsartikel (refereegranskat)abstract
- Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.
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| 5. |
- Hörnberg, Andreas, et al.
(författare)
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A comparative analysis of 23 structures of the amyloidogenic protein transthyretin.
- 2000
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Ingår i: J Mol Biol. - 0022-2836. ; 302:3, s. 649-69
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Tidskriftsartikel (refereegranskat)abstract
- Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.
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| 6. |
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| 7. |
- Hörnberg, Andreas, 1972-, et al.
(författare)
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The effect of iodide and chloride on transthyretin structure and stability
- 2005
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:26, s. 9290-9299
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.
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| 8. |
- Hörnberg, Andreas, et al.
(författare)
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The β-strand D of transthyretin trapped in two discrete conformations
- 2004
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Ingår i: Biochimica et Biophysica Acta. - Amsterdam : Elsevier. - 0006-3002 .- 1878-2434. ; 1700:1, s. 93-104
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Tidskriftsartikel (refereegranskat)abstract
- Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.
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| 9. |
- Hörnberg, Andreas, 1972-
(författare)
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Transthyretin from a structural perspective
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation.Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.
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