| 1. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 2. |
- Horsefield, Rob, 1977, et al.
(författare)
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High-resolution x-ray structure of human aquaporin 5
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:36, s. 13327-13332
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 5 (HsAQP5)facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-angstrom resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.
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| 3. |
- Grāve, Kristīne, 1988-
(författare)
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Structural basis for metalloprotein catalysis : Characterization of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase PgsA1 and Bacillus anthracis ribonucleotide reductase R2
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- About a third of all proteins need to associate with a particular metal ion or metallo-inorganic cofactor to function. This interplay expands the catalytic repertoire of enzymes and reflects the adaption of these catalytic macromolecules to the environments they have evolved in. A large portion of this work focuses on the membrane metalloprotein PgsA1 from the pathogen Mycobacterium tuberculosis and a radical-harboring protein R2 from the pathogen Bacillus anthracis, offering a glimpse into the metalloprotein universe and the catalysis they perform.This thesis is divided into two parts; the first part describes a method for high-throughput M. tuberculosis membrane protein expression screening in Escherichia coli and Mycobacterium smegmatis. This method employs target membrane protein fusions with the folding reporter Green Fluorescent Protein, allowing for fast selection of well-expressing membrane protein targets for further structural and functional characterization. This technique allowed overexpression of M. tuberculosis phosphatidylinositol phosphate synthase PgsA1, leading to its crystallization and the characterization of its high-resolution three-dimensional structure. PgsA1 is a MgII- dependent enzyme, catalyzing a vital step in the biosynthesis of phosphatidylinositol – one of the major phospholipids comprising the complex mycobacterial cell envelope. Therefore, PgsA1 presents an attractive target for the development of new antibiotics against tuberculosis.The second part of this thesis concerns the structural characterization of the B. anthracis class Ib ribonucleotide reductase radical-generating subunit R2 (R2b). R2b contains a dinuclear metallocofactor, which is able to be activated by dioxygen and generates a stable tyrosyl radical; the radical is further used for initiation of nucleotide reduction in the catalytic subunit of ribonucleotide reductase. R2b proteins utilize a di-manganese cofactor in vivo, but can also generate the radical using a di-iron cofactor in vitro, albeit less efficiently. How does R2b achieve correct metallation for efficient catalysis? We show that the B. anthracis R2b protein scaffold is able to select manganese over iron, and furthermore, describe the structural features that govern this metal-specificity. In addition, we describe redox-dependent structural changes in di-iron B. anthracis R2b after reaction with O2, and propose their role in gating solvent access to the metallocofactor and the radical site.
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| 4. |
- Hedfalk, Kristina, et al.
(författare)
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Aquaporin gating
- 2006
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Ingår i: Current Opinion in Structural Biology. - : Elsevier BV. - 1879-033X .- 0959-440X. ; 16, s. 447-456
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Forskningsöversikt (refereegranskat)abstract
- An acceleration in the rate at which new aquaporin structures are determined means that structural models are now available for mammalian AQP0, AQP1, AQP2 and AQP4, bacterial GlpF, AqpM and AQPZ, and the plant SoPIP2;1. With an apparent consensus emerging concerning the mechanism of selective water transport and proton extrusion, emphasis has shifted towards the issues of substrate selectivity and the mechanisms of aquaporin regulation. In particular, recently determined structures of plant SoPIP2;1, sheep and bovine AQP0, and Escherichia coli AQPZ provide new insights into the underlying structural mechanisms by which water transport rates are regulated in diverse organisms. From these results, two distinct pictures of 'capping' and 'pinching' have emerged to describe aquaporin gating.
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