| 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. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris.
- 2009
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 26:4, s. 215-27
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.
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| 3. |
- Backmark, Anna, 1979
(författare)
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Structural Studies of the Human Water Channel Protein Aquaporin 5
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aquaporins are water channel proteins found in the membranes of all living cells. In humans, thirteen aquaporins have been identified to date. Many of them have been implicated in water imbalance disorders. Water transport through the aquaporins is regulated at several levels. Regulation by trafficking involves translocation of aquaporin containing vesicles from intracellular storage sites to the cell membrane in response to cellular stimuli and after protein phosphorylation. Protein trafficking is a universal mechanism of great importance for cellularfunction.This thesis presents the X-ray structure of human aquaporin 5 (AQP5). It is the first structureof a human aquaporin with enough detail to detect structural features unique to the aquaporinhomologues in man. The structure of AQP5 presents new information regarding the functional role of the putative tetrameric pore of aquaporins. Contradictory to suggestions of a transport function for this pore in other aquaporins, a lipid molecule completely occludes the pore in the centre of the AQP5 tetramer. The structure of AQP5 further provides insights into structural events that may signal the protein for trafficking. Four potential phosphorylationsites are revealed in the structure.The structure of AQP5 was solved using two constructs, one of which included a C-terminal affinity tag (His-tag). Comparison of aquaporin structures with and without a His-tag indicated that the tag influences crystal packing. The tag was found to reduce the diffractionresolution. However, the tag introduced additional spacing and asymmetry in the crystal packing, which reduced or prevented merohedral twinning.
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| 4. |
- 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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