| 1. |
- Molina, Daniel Martinez, et al.
(författare)
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Expression and purification of the recombinant membrane protein YidC : A case studyfor increased solubility and stability
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 62:1, s. 49-52
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Tidskriftsartikel (refereegranskat)abstract
- YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, trans- location and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 lg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high con- centrations and that was stable for weeks at +4 C. The isolated protein is thus well suited for structural studies.
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| 2. |
- Niegowski, Damian, 1978-, et al.
(författare)
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A simple strategy towards membrane protein purification and crystallization
- 2006
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 39:1-3, s. 83-87
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Tidskriftsartikel (refereegranskat)abstract
- A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.
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| 3. |
- Niegowski, Damian, 1978-, et al.
(författare)
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Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution
- 2006
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 313:5785, s. 354-357
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Tidskriftsartikel (refereegranskat)abstract
- CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2 transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone– shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.
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| 4. |
- Niegowski, Damian, 1978-, et al.
(författare)
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Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 448:7153, s. 613-616
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Tidskriftsartikel (refereegranskat)abstract
- Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase1: this eaction is the key step in cysteinyl leukotriene formation. Here we present the rystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A ̊resolution, respectively. The structure reveals a homotrimer, here each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a orseshoe-shaped conformation on GSH, and effectively positions the thiol group or activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the v-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the eactive epoxide at the thiol of glutathione. This provides new structural insights nto the mechanism of LTC4 formation, and also suggests that the observed inding and activation of GSH might be common for a family of homologous proteins mportant for inflammatory and detoxification responses.
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| 5. |
- Niegowski, Damian, 1978-
(författare)
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Structural biology of integral membrane proteins - From methods to molecular mechanisms
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure. Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials.Despite the challenges in membrane protein structure determination two structures are presented in the thesis:The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway.The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4.
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| 6. |
- Niegowski, Damian, et al.
(författare)
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The CorA family : Structure and function revisited
- 2007
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 64:19-20, s. 2564-2574
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Forskningsöversikt (refereegranskat)abstract
- The CorA family is a group of ion transporters that mediate transport of divalent metal ions across biological membranes. Metal ions are essential elements in most cellular processes and hence the concentrations of ions in cells and organelles must be kept at appropriate levels. Impairment of these systems is implied in a number of pathological conditions. CorA proteins are abundant among the prokaryotic organisms but homologues are present in both human and yeast. The activity of CorA proteins has generally been associated with the transport of magnesium ions but the members of the CorA family can also transport other ions such as cobalt and nickel. The structure of the CorA from Thermotoga maritima, which also was the first structure of a divalent cation transporter determined, has opened the possibilities for understanding the mechanisms behind the ion transport and also corrected a number of assumptions that have been made in the past.
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| 7. |
- Stsiapanava, Alena, et al.
(författare)
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Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.
- 2014
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1844:2, s. 439-446
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Tidskriftsartikel (refereegranskat)abstract
- Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.
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